Compositions and methods for inhibiting expression of a gene from the jc virus

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a gene from the JC Virus (JC virus genome), comprising an antisense strand having a nucleotide sequence which is less that 30 nucleotides in length, generally 19-25 nucleotides in length, and which is substantially complementary to at least a part of a gene from the JC Virus. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by JC virus expression and the expression of a gene from the JC Virus using the pharmaceutical composition; and methods for inhibiting the expression of a gene from the JC Virus in a cell.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/795,765, filed Apr. 28, 2006, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to inhibit the expression of one of the genes of the JC virus and the use of the dsRNA to treat pathological processes mediated by JC virus infection, such as PML.

BACKGROUND OF THE INVENTION

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system which results from reactivation of the latent polyomavirus JC virus (JCV) and its productive replication in glial cells of the human brain (Berger, J. R. (1995) J. Neurovirol. 1:5-18). Once a rare disease primarily seen in patients with impaired immune systems due to lymphoproliferative and myeloproliferative disorders, PML has become one of the major neurologic problems among patients with AIDS (Cinque, P., (2003). J. Neurovirol. 9(Suppl. 1):88-92).

It has been reported that between 4 and 8% of AIDS patients exhibit signs of PML, and JCV has been detected in the cerebrospinal fluid of affected patients, suggesting that there is active replication of the virus in the brain (Berger, J. R. (1995) J. Neurovirol. 1:5-18, Clifford, D. B., (2001) J. Neurovirol. 4:279). In addition, PML has recently been seen in patients undergoing experimental treatment with Tsybari, an anti VLA4 antobody, in combination with interferon. The histological hallmarks of PML include multifocal demyelinated lesions with enlarged eosinophilic nuclei in oligodendrocytes and enlarged bizarre astrocytes with lobulated hyperchromatic nuclei within white matter tracts of the brain (Cinque, P., (2003). J. Neurovirol. 9(Suppl. 1):88-92), although in some instances atypical features that include a unifocal pattern of demyelination and involvement of the gray matter have been reported (Sweeney, B. J., (1994). J. Neurol. Neurosurg. Psychiatry 57:994-997). Earlier observations from in vitro cell culture studies and an in vivo evaluation of JCV in clinical samples led to early assumptions that oligodendrocytes and astrocytes are the only cells which support productive viral infections (Gordon, J. (1998) Int. J. Mol. Med. 1:647-655). Accordingly, molecular studies have provided evidence for cell-type-specific transcription of the viral early genome in cells derived from the central nervous system (Raj, G. V., (1995) Virology 10:283-291). However, subsequent studies have shown low, but detectable, levels of JCV gene expression in nonneural cells, including B cells, and noticeably high levels of production of the viral early protein in several neural and nonneural tumor cells in humans (Gordon, J. (1998) Int. J. Mol. Med. 1:647-655, Khalili, K., 2003. Oncogene 22:5181-5191).

Like the other polyomaviruses, JCV is a small DNA virus whose genome can be divided into three regions that encompass the transcription control region; the genes responsible for the expression of the viral early protein, T antigen; and the genes encoding the viral late proteins, VP1, VP2, and VP3. In addition, the late genome is also responsible for production of an auxiliary viral protein, agnoprotein. T-antigen expression is pivotal for initiation of the viral lytic cycle, as this protein stimulates transcription of the late genes and induces the process of viral DNA replication. Recent studies have ascribed an important role for agnoprotein in the transcription and replication of JCV, as inhibition of its production significantly reduced viral gene expression and replication (M. Safak et al., unpublished observations). Furthermore, the agnoprotein dysregulates the cell cycle by altering the expression of several cyclins and their associated kinases (Darbinyan, A., (2002) Oncogene 21:5574-5581).

Thus far, there are no effective therapies for the suppression of JCV replication and the treatment of PML. Cytosine arabinoside (AraC) has been tested for the treatment of PML patients, and the outcome in some instances revealed a remission of JCV-associated demyelination (Aksamit, A. (2001) J. Neurovirol. 7:386-390). Reports from the AIDS Clinical Trial Group Organized Trial 243, however, have suggested that there is no difference in the survival of human immunodeficiency virus type 1 (HIV-1)-infected patients with PML and that of the control population, although in other reports it has been suggested that the failure of AraC in the AIDS Clinical Trial Group trial may have been due to insufficient delivery of the AraC via the intravenous and intrathecal routes (Levy, R. M., (2001) J. Neurovirol. 7:382-385). Based on in vitro studies showing the ability of inhibitors of topoisomerase to suppress JCV DNA replication, the topoisomerase inhibitor topotecan was used for the treatment of AIDS-PML patients, and the results suggested that topotecan treatment may be associated with a decreased lesion size and prolonged survival (Royal, W., III, (2003) J. Neurovirol. 9:411-419).

Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.

Recent reports have indicated that in vitro, RNAi may show some promise in reducing JC virus replication (Radhakrishnan, S. (2004) J. Vir. 78:7264-7269, Orba, Y. (2004) J. Vir. 78:7270-7273). However, the RNAi agents examined were not designed against all know JC Virus strains and were not selected for stability and other properties need for in vivo therapeutic RNAi agents. Accordingly, despite significant advances in the field of RNAi, there remains a need for an agent that can selectively and efficiently silence a gene in the JC virus using the cell's own RNAi machinery that has both high biological activity and in vivo stability, and that can effectively inhibit replication of the JC virus for use in treating pathological processes mediated by JC virus infection.

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the JC virus in a cell or mammal using such dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases caused by JC viral infection, such as PML. The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of a gene from the JC Virus.

In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression one of the genes of the JC virus and viral replication. The dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a nucleotide sequence which is substantially complementary to at least part of an mRNA encoded by a gene from the JC Virus, and the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length. The dsRNA, upon contacting with a cell expressing infected with the JC virus, inhibits the expression of a gene from the JC Virus by at least 40%.

For example, the dsRNA molecules of the invention can be comprised of a first sequence of the dsRNA that is selected from the group consisting of the sense sequences of Tables 1a and b and the second sequence is selected from the group consisting of the antisense sequences of Tables 1a and b. The dsRNA molecules of the invention can be comprised of naturally occurring nucleotides or can be comprised of at least one modified nucleotide, such as a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative. Alternatively, the modified nucleotide may be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. Generally, such modified sequence will be based on a first sequence of said dsRNA selected from the group consisting of the sense sequences of Tables 1a and b and a second sequence selected from the group consisting of the antisense sequences of Tables 1a and b.

In another embodiment, the invention provides a cell comprising one of the dsRNAs of the invention. The cell is generally a mammalian cell, such as a human cell.

In another embodiment, the invention provides a pharmaceutical composition for inhibiting the replication of the JC virus in an organism, generally a human subject, comprising one or more of the dsRNA of the invention and a pharmaceutically acceptable carrier or delivery vehicle.

In another embodiment, the invention provides a method for inhibiting the expression of a gene in the JC Virus in a cell, comprising the following steps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid         (dsRNA), wherein the dsRNA comprises at least two sequences that         are complementary to each other. The dsRNA comprises a sense         strand comprising a first sequence and an antisense strand         comprising a second sequence. The antisense strand comprises a         region of complementarity which is substantially complementary         to at least a part of a mRNA encoded by the JC virus, and         wherein the region of complementarity is less than 30         nucleotides in length, generally 19-24 nucleotides in length,         and wherein the dsRNA, upon contact with a cell infected with         the JC virus, inhibits expression of a gene from the JC Virus by         at least 40%; and     -   (b) maintaining the cell produced in step (a) for a time         sufficient to obtain degradation of the mRNA transcript of a JC         virus gene, thereby inhibiting expression of a gene from the JC         Virus in the cell.

In another embodiment, the invention provides methods for treating, preventing or managing pathological processes mediated by JC virus infection, e.g. such as PML, comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs of the invention.

In another embodiment, the invention provides vectors for inhibiting the expression of a gene of the JC virus in a cell, comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.

In another embodiment, the invention provides a cell comprising a vector for inhibiting the expression of a gene of the JC virus in a cell. The vector comprises a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.

BRIEF DESCRIPTION OF THE FIGURES

No Figures are presented.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of a gene from the JC Virus in a cell or mammal using the dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by JC virus infection using dsRNA. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).

The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of a gene from the JC Virus. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in replication and or maintenance of JC virus infection and the occurrence of PML in a subject infected with the JC virus. Using cell-based and animal assays, the present inventors have demonstrated that very low dosages of these dsRNA can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a gene from the JC Virus. Thus, the methods and compositions of the invention comprising these dsRNAs are useful for treating pathological processes mediated by JC viral infection, e.g. cancer, by targeting a gene involved in JC virus relication and/or maintenance in a cell.

The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of a gene from the JC virus, as well as compositions and methods for treating diseases and disorders caused by the infection with the JC virus, such as PML. The pharmaceutical compositions of the invention comprise a dsRNA having an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of a gene from the JC Virus, together with a pharmaceutically acceptable carrier.

Accordingly, certain aspects of the invention provide pharmaceutical compositions comprising the dsRNA of the invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of a gene in a gene from the JC Virus, and methods of using the pharmaceutical compositions to treat diseases caused by infection with the JC virus.

I. Definitions

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.

As used herein, “JC virus” refers to the latent polyomavirus JC Virus that has a reference sequence NC_(—)001699. In addition, further accession numbers of various JCVirus sequences are AB038249.1-AB038255.1, AB048545.1-AB048582.1, AB074575.1-AB074591.1, AB077855.1-AB077879.1, AB081005.1-AB081030.1, AB081600.1-AB081618.1, AB081654.1, AB092578.1-AB092587.1, AB103387.1, AB103402.1-AB103423.1, AB104487.1, AB113118.1-AB113145.1, AB118651.1-AB118659.1, AB126981.1-AB127027.1, AB127342.1, AB127344.1, AB127346.1-AB127349.1, AB127352.1-AB127353.1, AB198940.1-AB198954.1, AB220939.1-AB220943.1, AF004349.1-AF004350.1, AF015526.1-AF015537.1, AF015684.1, AF030085.1, AF281599.1-AF281626.1, AF295731.1-AF295739.1, AF300945.1-AF300967.1, AF363830.1-AF363834.1, AF396422.1-AF396435.1, AY121907.1-AY121915.1, NC_(—)001699.1, U61771.1, U73500.1-U73502.1.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene from the JC Virus, including mRNA that is a product of RNA processing of a primary transcription product.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes of the invention.

“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.

The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding JC virus). For example, a polynucleotide is complementary to at least a part of a JC virus mRNA if the sequence is substantially complementary to a non-interrupted portion of a mRNA encoding JC virus.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs.

As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

“Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.

The terms “silence” and “inhibit the expression of”, in as far as they refer to a gene from the JC Virus, herein refer to the at least partial suppression of the expression of a gene from the JC Virus, as manifested by a reduction of the amount of mRNA transcribed from a gene from the JC Virus which may be isolated from a first cell or group of cells in which a gene from the JC Virus is transcribed and which has or have been treated such that the expression of a gene from the JC Virus is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to JC virus genome transcription, e.g. the amount of protein encoded by a gene from the JC Virus, or the number of cells displaying a certain phenotype, e.g infection with the JC Virus. In principle, JC virus genome silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of a gene from the JC Virus by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.

For example, in certain instances, expression of a gene from the JC Virus is suppressed by at least about 20%, 25%, 35%, or 50% by administration of the double-stranded oligonucleotide of the invention. In some embodiment, a gene from the JC Virus is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In some embodiments, a gene from the JC Virus is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention.

As used herein in the context of JC virus expression, the terms “treat”, “treatment”, and the like, refer to relief from or alleviation of pathological processes mediated by JC virus infection. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by JC virus expression), the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.

As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by JC virus infection or an overt symptom of pathological processes mediated by JC virus expression. The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of pathological processes mediated by JC virus infection, the patient's history and age, the stage of pathological processes mediated by JC virus infection, and the administration of other anti-pathological processes mediated by JC virus infection.

As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.

II. Double-Stranded Ribonucleic Acid (dsRNA)

In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a gene from the JC Virus in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a gene from the JC Virus, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein said dsRNA, upon contact with a cell expressing the gene from the JC virus, inhibits the expression of the JC virus gene by at least 40%.

The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a gene from the JC Virus, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. The dsRNA of the invention may further comprise one or more single-stranded nucleotide overhang(s).

The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In a preferred embodiment, a gene from the JC Virus is the human JC virus genome. In specific embodiments, the antisense strand of the dsRNA comprises the sense sequences of Tables 1a and b and the second sequence is selected from the group consisting of the antisense sequences of Tables 1a and b. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 1a and b can readily be determined using the target sequence and the flanking JC virus sequence.

In further embodiments, the dsRNA comprises at least one nucleotide sequence selected from the groups of sequences provided in Tables 1a and b. In other embodiments, the dsRNA comprises at least two sequences selected from this group, wherein one of the at least two sequences is complementary to another of the at least two sequences, and one of the at least two sequences is substantially complementary to a sequence of an mRNA generated in the expression of a gene from the JC Virus. Generally, the dsRNA comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in Tables 1a and b and the second oligonucleotide is described as the antisense strand in Tables 1a and b The skilled person is well aware that dsRNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 1a and b, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter dsRNAs comprising one of the sequences of Tables 1a and b minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1a and b, and differing in their ability to inhibit the expression of a gene from the JC Virus in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further dsRNAs that cleave within the target sequence provided in Tables 1a and b can readily be made using the JC virus sequence and the target sequence provided.

In addition, the RNAi agents provided in Tables 1a and b identify a site in the JC virus mRNA that is susceptible to RNAi based cleavage. As such the present invention further includes RNAi agents that target within the sequence targeted by one of the agents of the present invention. As used herein a second RNAi agent is said to target within the sequence of a first RNAi agent if the second RNAi agent cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent. Such a second agent will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 1a and b coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a gene from the JC Virus. For example, the last 15 nucleotides of SEQ ID NO:1 combined with the next 6 nucleotides from the target JC virus genome produces a single strand agent of 21 nucleotides that is based on one of the sequences provided in Tables 1a and b.

The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of a gene from the JC Virus, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a gene from the JC Virus. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of a gene from the JC Virus is important, especially if the particular region of complementarity in a gene from the JC Virus is known to have polymorphic sequence variation within the population.

In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA may also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Specific examples of preferred dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Preferred modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference

Preferred modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.

In other preferred dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH.sub.2-NH—CH.sub.2-, —CH.sub.2-N(CH.sub.3)-O—CH.sub.2-[known as a methylene (methylimino) or MMI backbone], —CH.sub.2-O—N(CH.sub.3)-CH.sub.2-, —CH.sub.2-N(CH.sub.3)-—N(CH.sub.3)-CH.sub.2- and —N(CH.sub.3)-CH.sub.2-CH.sub.2-[wherein the native phosphodiester backbone is represented as—O—P—O—CH.sub.2-] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub. 1 to C.sub. 10 alkyl or C.sub.2 to C.sub. 10 alkenyl and alkynyl. Particularly preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C.sub. 1 to C.sub. 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy(2′-O—CH.sub.2CH.sub.2OCH.sub.3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH.sub.2-O—CH.sub.2-N(CH.sub.2).sub.2, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy(2′-OCH.sub.3), 2′-aminopropoxy(2′-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

dsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.

Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994 4 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compounds or “chimeras,” in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.

Vector Encoded RNAi Agents

The dsRNA of the invention can also be expressed from recombinant viral vectors intracellularly in vivo. The recombinant viral vectors of the invention comprise sequences encoding the dsRNA of the invention and any suitable promoter for expressing the dsRNA sequences. Suitable promoters include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the dsRNA in a particular tissue or in a particular intracellular environment. The use of recombinant viral vectors to deliver dsRNA of the invention to cells in vivo is discussed in more detail below.

dsRNA of the invention can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g, lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.

For example, lentiviral vectors of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

III. Pharmaceutical Compositions Comprising dsRNA

In one embodiment, the invention provides pharmaceutical compositions comprising a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition comprising the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a gene from the JC Virus and/or viral infection, such as PML. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery.

The pharmaceutical compositions of the invention are administered in dosages sufficient to inhibit expression of a gene from the JC Virus. The present inventors have found that, because of their improved efficiency, compositions comprising the dsRNA of the invention can be administered at surprisingly low dosages. A maximum dosage of 5 mg dsRNA per kilogram body weight of recipient per day is sufficient to inhibit or completely suppress expression of a gene from the JC Virus.

In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 microgram to 1 mg per kilogram body weight per day. The pharmaceutical composition may be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by JC virus expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose.

The present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Liposomes

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G.sub.M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G.sub.M1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C.sub.1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an dsRNA RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNA dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Agents that enhance uptake of dsRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphor-amide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulation a range of dosage for use in humans. The dosage of compositions of the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In addition to their administration individually or as a plurality, as discussed above, the dsRNAs of the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by JC virus expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Methods for Treating Diseases Caused by Expression of a Gene from the JC Virus

The invention relates in particular to the use of a dsRNA or a pharmaceutical composition prepared therefrom for the treatment or prevention of pathological conditions associated with JC Virus infection, e.g., PML. Owing to the inhibitory effect on JC virus expression, an dsRNA according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life, particularly in a patient being treated with an anti-VLA4 antibody as part of treatment for MS.

The invention furthermore relates to the use of an dsRNA or a pharmaceutical composition thereof for treating PML in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating cancer and/or for preventing tumor metastasis. Preference is given to a combination with radiation therapy and chemotherapeutic agents, such as cisplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

The invention can also be practiced by including with a specific RNAi agent, in combination with another anti-cancer chemotherapeutic agent, such as any conventional chemotherapeutic agent. The combination of a specific binding agent with such other agents can potentiate the chemotherapeutic protocol. Numerous chemotherapeutic protocols will present themselves in the mind of the skilled practitioner as being capable of incorporation into the method of the invention. Any chemotherapeutic agent can be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, as well as natural products. For example, the compound of the invention can be administered with antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH). Other antineoplastic protocols include the use of a tetracycline compound with another treatment modality, e.g., surgery, radiation, etc., also referred to herein as “adjunct antineoplastic modalities.” Thus, the method of the invention can be employed with such conventional regimens with the benefit of reducing side effects and enhancing efficacy.

Methods for Inhibiting Expression of a Gene from the JC Virus

In yet another aspect, the invention provides a method for inhibiting the expression of a gene from the JC Virus in a mammal. The method comprises administering a composition of the invention to the mammal such that expression of the target JC virus genome is silenced. Because of their high specificity, the dsRNAs of the invention specifically target RNAs (primary or processed) of the target JC virus gene. Compositions and methods for inhibiting the expression of these JC virus genes using dsRNAs can be performed as described elsewhere herein.

In one embodiment, the method comprises administering a composition comprising a dsRNA, wherein the dsRNA comprises a nucleotide sequence which is complementary to at least a part of an RNA transcript of a gene from the JC Virus, to the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, administration. In preferred embodiments, the compositions are administered by intravenous infusion or injection.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES Design of JCV siRNAs

Full-length genome sequences to JC virus available on Apr. 10, 2006, were obtained, resulting in a target pool of 388 sequences (accession numbers: AB038249.1-AB038255.1; AB048545.1-AB048582.1; AB074575.1-AB074591.1; AB077855.1-AB077879.1; AB081005.1-AB081030.1; AB081600.1-AB081618.1; AB081654.1; AB092578.1-AB092587.1; AB103387.1; AB103402.1-AB103423.1; AB104487.1; AB113118.1-AB113145.1; AB118651.1-AB118659.1; AB126981.1-AB127027.1; AB127342.1; AB127344.1; AB127346.1-AB127349.1; AB127352.1-AB127353.1; AB198940.1-AB198954.1; AB220939.1-AB220943.1; AF004349.1-AF004350.1; AF015526.1-AF015537.1; AF015684.1; AF030085.1; AF281599.1-AF281626.1; AF295731.1-AF295739.1; AF300945.1-AF300967.1; AF363830.1-AF363834.1; AF396422.1-AF396435.1; AY121907.1-AY121915.1; NC_(—)001699.1; U61771.1; U73500.1-U73502.1). NC_(—)001699 was defined as reference sequence.

The siRNA selection process was run as follows: ClustalW multiple alignment was used to generate a global alignment of all sequences from the target pool. An IUPAC consensus sequence was then generated.

All conserved 19mer target sequences from the IUPAC consensus represented by stretches containing only A, T, C or G bases, which are therefore present in all sequences of the target pool were selected. In order to only select siRNAs that target transcribed sequence parts of the JC virus, candidate target sequences were selected out of the pool of conserved 19mer target sequences. For this, candidate target sequences covering regions between nucleotide 163-2594 and between 2527-5115 relative to reference sequence were extracted for late and early genes, respectively. Further, as sequences for early genes are in reverse complement orientation compared with genomic sequences, candidate target sequences of these genes were transferred to reverse complement sequences and replaced the former pool of candidate target sequences.

In order to rank candidate target sequences and their respective siRNAs and select appropriate ones, their predicted potential for interacting with irrelevant targets (off-target potential) was taken as a ranking parameter. siRNAs with low off-target potential were defined as preferable and assumed to be more specific in vivo.

For predicting siRNA-specific off-target potential, the following assumptions were made:

-   -   1) positions 2 to 9 (counting 5′ to 3′) of a strand (seed         region) may contribute more to off-target potential than rest of         sequence (non-seed and cleavage site region)     -   2) positions 10 and 11 (counting 5′ to 3′) of a strand (cleavage         site region) may contribute more to off-target potential than         non-seed region     -   3) an off-target score can be calculated for each hit, based on         identity to siRNA sequence and position of mismatches     -   4) assuming potential abortion of sense strand activity by         internal modifications introduced, only off-target potential of         antisense strand will be relevant

To identify potential off-target genes, 19mer input sequences were subjected to a homology search against publicly available human mRNA sequences.

To this purpose, fastA (version 3.4) searches were performed with all 19mer candidate target sequences against a human RefSeq database (downloaded available version from ftp://ftp.ncbi.nih.gov/refseq/ on Nov. 7, 2006). FastA searches were executed with parameters-values-pairs-f 50-g 50 in order to take into account the homology over the full length of the 19mer without any gaps. In order to ensure the listing of all relevant off-target hits in the fastA output file the parameter-E 30000 was used in addition. A scoring matrix was applied for the run that assessed every nucleotide match with a score of 13 and every mismatch with a score of −7. The search resulted in a list of potential off-targets for each candidate siRNA.

To sort the resulting list of potential off-targets for each siRNA, fastA output files were analyzed to identify the best off-target and its off-target score. The following off-target properties for each 19mer input sequence were extracted for each off-target to calculate the off-target score:

Number of mismatches in non-seed region

Number of mismatches in seed region

Number of mismatches in cleavage site region

The off-target score was calculated for considering assumption 1 to 3 as follows:

Off-target score=number of seed mismatches*10+number of cleavage site mismatches*1.2+number of non-seed mismatches*1

The most relevant off-target gene for input each 19mer input sequences was defined as the gene with the lowest off-target score. Accordingly, the lowest off-target score was defined as the relevant off-target score for the corresponding siRNA.

In order to generate a ranking for siRNAs, calculated relevant off-target scores were transferred into a result table. All siRNAs were sorted according to the off-target score (descending).

An off-target score of 2.2 was defined as cut-off for siRNA selection (specificity criterion). In addition, all sequences with only one mismatch in the seed region were eliminated from the screening set. The selection procedure resulted in a set of 93 JCV specific siRNAs (Table 1a).

An expanded screening was generated by re-calculating the predicted specificity based on the newly available human RefSeq database (Human mRNA sequences in RefSeq release version 21 (downloaded Jan. 12, 2007)) and selecting only 208 siRNAs that did not contain more than 3 G's in a row and had an off-target score of at least 2 for the antisense strand (Table 1b).

Synthesis of JCV siRNAs

All siRNAs were synthesized in 0.2 μmole synthesis scale on an ABI3900 DNA synthesizer according to standard procedures.

For the initial screening set (93 different siRNA sequences), 4 different strategies of chemical modification were used:

-   a) exo/endo light (EEL): —sense strand: 2′-O-methyl @ all     pyrimidines, PTO between nucleotides 20 and 21 (counting from     5′-end), dTdT at 3′-end (nucleotides 20 and 21)     -   —antisense strand: 2′-O-methyl at pyrimidines in 5′-UA-3′ and         5′-CA-3′ motives, PTO between nucleotides 20 and 21 (counting         from 5′-end), dTdT at 3′-end (nucleotides 20 and 21)

b) EEL plus 2′-O-methyl in position 2 of antisense strand (only if no 5′-UA-3′ and 5′-CA-3′ at 5′-end, otherwise already covered by EEL)

c) EEL plus 2′-O-methyl in position 2 of sense strand (only if no pyrimidine in position 2, otherwise already covered by EEL)

d) EEL plus 2′-O-methyl in position 2 of sense and antisense strand (only if not already covered by a, b, and c) (Table 1a)

For the expanded screening set (208 different siRNA sequences), siRNAs were composed of unmodified RNA oligonucleotides with dT/dT overhangs (dTdT at 3′-end (nucleotides 20 and 21) of antisense and sense strands) (Table 1b).

Screening of JCV siRNAs

Construction of Reporter-Systems Encoding JCV Transcripts

The sequence of the early JCV transcript (E) was synthesized at GENEART (Regensburg, Germany) and cloned into GENEART standard vectors. The sequence of the late JCV transcript was subdivided in a first approach into two fragments: L1, including the transcript sequence of the VP1 protein, and LA23, including the sequences of VP2, VP3 and the Agnoprotein. Due to cloning problems with fragment LA23, this sequence was subdivided in a second approach into two fragments (LA23 1-700 and LA23 701-1438). All sequences were synthesized at GENEART and cloned into GENEART standard vectors. All fragments (E, L1, LA23 1-700 and LA23 701-1438) were subcloned into psiCheck-2 (Promega, Mannheim, Germany) via XhoI and NotI (both NEB, Frankfurt, Germany), resulting in constructs with the JCV sequences between the stop-codon and the polyA-signal of Renilla luciferase.

L1 (SEQ ID NO:931) CTCGAGACTTTTAGGGTTGTACGGGACTGTAACACCTGCTCTTGAAGCAT ATGAAGATGGCCCCAACAAAAAGAAAAGGAGAAAGGAAGGACCCCGTGCA AGTTCCAAAACTTCTTATAAGAGGAGGAGTAGAAGTTCTAGAAGTTAAAA CTGGGGTTGACTCAATTACAGAGGTAGAATGCTTTTTAACTCCAGAAATG GGTGACCCAGATGAGCATCTTAGGGGTTTTAGTAAGTCAATTTCTATATC AGATACATTTGAAAGTGACTCCCCAAATAAGGACATGCTTCCTTGTTACA GTGTGGCCAGAATTCCACTACCCAATCTAAATGAGGATCTAACCTGTGGA AATATACTAATGTGGGAGGCTGTGACCTTAAAAACTGAGGTTCTAGGGGT GACAACTTTGATGAATGTGCACTCTAATGGTCAAGCAACTCATGACAATG GTGCAGGAAAGCCAGTGCAGGGCACCAGCTTTCATTTTTTTTCTGTTGGC GGGGAGGCTTTAGAATTACAGGGGGTGGTTTTTAATTACAGAACAAAGTA CCCAGATGGAACAATTTTTCCAAAGAATGCAACAGTGCAATCTCAAGTAA TGAACACAGAGCACAAGGCGTACCTAGATAAGAACAAAGCATATCCTGTT GAATGTTGGGTTCCTGATCCCACCAGAAATGAAAACACAAGATATTTTGG GACACTAACAGGAGGAGAAAATGTTCCTCCAGTTCTTCATATAACAAACA CTGCCACAACAGTGCTGCTTGATGAATTTGGTGTTGGGCCACTTTGCAAA GGTGACAACTTGTATTTGTCAGCTGTTGATGTTTGTGGAATGTTTACTAA CAGATCTGGTTCCCAGCAGTGGAGAGGACTGTCCAGATATTTTAAGGTTC AGCTCAGAAAAAGGAGGGTTAAAAACCCCTACCCAATTTCTTTCCTTCTT ACTGATTTGATTAACAGAAGGACCCCTAGAGTTGATGGGCAACCTATGTA TGGTATGGATGCTCAGGTAGAGGAGGTTAGAGTTTTTGAGGGGACAGAGG AACTTCCAGGGGACCCAGACATGATGAGATATGTTGACAGATATGGACAG TTGCAAACAAAGATGCTGTAATCAAAATCCTTTATTGTAATATGCAGTAC ATTTTAATAAAGTATAACCAGCTTTACTTTACAGTTGCAGTCATGCGGCC GC E (SEQ ID NO:932) CTCGAGCCGCCTCCAAGCTTACTCAGAAGTAGTAAGGGCGTGGAGGCTTT TTAGGAGGCCAGGGAAATTCCCTTGTTTTTCCCTTTTTTGCAGTAATTTT TTGCTGCAAAAAGCTAAAATGGACAAAGTGCTGAATAGGGAGGAATCCAT GGAGCTTATGGATTTATTAGGCCTTGATAGGTCTGCATGGGGGAACATTC CTGTCATGAGAAAAGCTTATCTGAAAAAATGCAAAGAACTCCACCCTGAT AAAGGTGGGGACGAAGACAAGATGAAGAGAATGAATTTTTTATATAAAAA AATGGAACAAGGTGTAAAAGTTGCTCATCAGCCTGATTTTGGTACATGGA ATAGTTCAGAGGTTGGTTGTGATTTTCCTCCTAATTCTGATACCCTTTAT TGCAAGGAATGGCCTAACTGTGCCACTAATCCTTCAGTGCATTGCCCCTG TTTAATGTGCATGCTAAAATTAAGGCATAGAAACAGAAAATTTTTAAGAA GCAGCCCACTTGTGTGGATAGATTGCTATTGCTTTGATTGCTTCAGACAA TGGTTTGGGTGTGACTTAACCCAAGAAGCTCTTCATTGCTGGGAGAAAGT TCTTGGAGACACCCCCTACAGGGATCTAAAGCTTTAAGTGCCAACCTATG GAACAGATGAATGGGAATCCTGGTGGAATACATTTAATGAGAAGTGGGAT GAAGACCTGTTTTGCCATGAAGAAATGTTTGCCAGTGATGATGAAAACAC AGGATCCCAACACTCTACCCCACCTAAAAAGAAAAAAAAGGTAGAAGACC CTAAAGACTTTCCTGTAGATCTGCATGCATTCCTCAGTCAAGCTGTGTTT AGTAATAGAACTGTTGCTTCTTTTGCTGTGTATACCACTAAAGAAAAAGC TCAAATTTTATATAAGAAACTTATGGAAAAATATTCTGTAACTTTTATAA GTAGACATGGTTTTGGGGGTCATAATATTTTGTTTTTCTTAACACCACAT AGACATAGAGTGTCAGCAATTAATAACTACTGTCAAAAACTATGTACCTT TAGTTTTTTAATTTGTAAAGGTGTGAATAAGGAATACTTGTTTTATAGTG CCCTGTGTAGACAGCCATATGCAGTAGTGGAAGAAAGTATTCAGGGGGGC CTTAAGGAGCATGACTTTAACCCAGAAGAACCAGAAGAAACTAAGCAGGT TTCATGGAAATTAGTTACACAGTATGCCTTGGAAACCAAGTGTGAGGATG TTTTTTTGCTTATGGGCATGTACTTAGACTTTCAGGAAAACCCACAGCAA TGCAAAAAATGTGAAAAAAAGGATCAGCCAAATCACTTTAACCATCATGA AAAACACTATTATAATGCCCAAATTTTTGCAGATAGCAAAAATCAAAAAA GCATTTGCCAGCAGGCTGTTGATACTGTAGCAGCCAAACAAAGGGTTGAC AGCATCCACATGACCAGAGAAGAAATGTTAGTTGAAAGGTTTAATTTCTT GCTTGATAAAATGGACTTAATTTTTGGGGCACATGGCAATGCTGTTTTAG AGCAATATATGGCTGGGGTGGCCTGGATTCATTGCTTGCTGCCTCAAATG GACACTGTTATTTATGACTTTCTAAAATGCATTGTATTAAACATTCCAAA AAAAAGGTACTGGCTATTCAAGGGGCCAATAGACAGTGGCAAAACTACTT TAGCTGCAGCTTTACTTGATCTCTGTGGGGGAAAGTCATTAAATGTTAAT ATGCCATTAGAAAGATTAAACTTTGAATTAGGAGTGGGTATAGATCAGTT TATGGTTGTATTTGAGGATGTAAAAGGCACTGGTGCAGAGTCAAGGGATT TACCTTCAGGGCATGGCATAAGCAACCTTGATTGCTTAAGAGATTACTTA GATGGAAGTGTAAAAGTTAATTTAGAGAGAAAACACCAAAACAAAAGAAC ACAGGTGTTTCCACCTGGAATTGTAACCATGAATGAATATTCAGTGCCTA GAACTTTACAGGCCAGATTTGTAAGGCAGATAGATTTTAGACCAAAGGCC TACCTGAGAAAATCACTAAGCTGCTCTGAGTATTTGCTAGAAAAAAGGAT TTTGCAAAGTGGTATGACTTTGCTTTTGCTTTTAATCTGGTTTAGGCCAG TTGCTGACTTTGCAGCTGCCATTCATGAGAGGATTGTGCAGTGGAAAGAA AGGCTGGATTTAGAAATAAGCATGTATACATTTTCTACTATGAAAGCTAA TGTTGGTATGGGGAGACCCATTCTTGACTTTCCTAGAGAGGAAGATTCTG AAGCAGAAGACTCTGGACATGGATCAAGCACTGAATCACAATCACAATGC TTTTCCCAGGTCTCAGAAGCCTCTGGTGCAGACACACAGGAAAACTGCAC TTTTCACATCTGTAAAGGCTTTCAATGTTTCAAAAAACCAAAGACCCCTC CCCCAAAATAACTGCAACTGTGCGGCCGC LA23 1-700 (SEQ ID NO:933) CTCGAGCAGCTAACAGCCAGTAAACAAAGCACAAGGGGAAGTGGAAAGCA GCCAAGGGAACATGTTTTGCGAGCCAGAGCTGTTTTGGCTTGTCACCAGC TGGCCATGGTTCTTCGCCAGCTGTCACGTAAGGCTTCTGTGAAAGTTAGT AAAACCTGGAGTGGAACTAAAAAAAGAGCTCAAAGGATTTTAATTTTTTT GTTAGAATTTTTGCTGGACTTTTGCACAGGTGAAGACAGTGTAGACGGGA AAAAAAGACAGAGACACAGTGGTTTGACTGAGCAGACATACAGTGCTTTG CCTGAACCAAAAGCTACATAGGTAAGTAATGTTTTTTTTTGTGTTTTCAG GTTCATGGGTGCCGCACTTGCACTTTTGGGGGACCTAGTTGCTACTGTTT CTGAGGCTGCTGCTGCCACAGGATTTTCAGTAGCTGAAATTGCTGCTGGA GAGGCTGCTGCTACTATAGAAGTTGAAATTGCATCCCTTGCTACTGTAGA GGGGATTACAAGTACCTCTGAGGCTATAGCTGCTATAGGCCTTACTCCTG AAACATATGCTGTAATAACTGGAGCTCCGGGGGCTGTAGCTGGGTTTGCT GCATTGGTTCAAACTGTAACTGGTGGTAGTGCTATTGCTCAGTTGGGATA TAGATTTTTTGCTGACTGGGATCATAAAGTTTCAACAGTTGGGCTTTTTC GCGGCCGC LA23 701-1438 (SEQ ID NO:934) CTCGAGAGCAGCCAGCTATGGCTTTACAATTATTTAATCCAGAAGACTAC TATGATATTTTATTTCCTGGAGTGAATGCCTTTGTTAACAATATTCACTA TTTAGATCCTAGACATTGGGGCCCGTCCTTGTTCTCCACAATCTCCCAGG CTTTTTGGAATCTTGTTAGAGATGATTTGCCAGCCTTAACCTCTCAGGAA ATTCAGAGAAGAACCCAAAAACTATTTGTTGAAAGTTTAGCAAGGTTTTT GGAAGAAACTACTTGGGCAATAGTTAATTCACCAGCTAACTTATATAATT ATATTTCAGACTATTATTCTAGATTGTCTCCAGTTAGGCCCTCTATGGTA AGGCAAGTTGCCCAAAGGGAGGGAACCTATATTTCTTTTGGCCACTCATA CACCCAAAGTATAGATGATGCAGACAGCATTCAAGAAGTTACCCAAAGGC TAGATTTAAAAACCCCAAATGTGCAATCTGGTGAATTTATAGAAAGAAGT ATTGCACCAGGAGGTGCAAATCAAAGATCTGCTCCTCAATGGATGTTGCC TTTACTTTTAGGGTTGTACGGGACTGTAACACCTGCTCTTGAAGCATATG AAGATGGCCCCAACAAAAAGAAAAGGAGAAAGGAAGGACCCCGTGCAAGT TCCAAAACTTCTTATAAGAGGAGGAGTAGAAGTTCTAGAAGTTAAAACTG GGGTTGACTCAATTACAGAGGTAGAATGCTGCGGCCGC

Screen of JCV siRNAs in Transfected Cells

Cos-7 cells (DSMZ, Braunschweig, Germany, # ACC-60) were seeded at 1.5×10⁴ cells/well on white 96-well plates with clear bottoms (Greiner Bio-One GmbH, Frickenhausen, Germany) in 75 μl of growth medium. Directly after seeding the cells, 50 ng of the corresponding reporter-plasmid per well was transfected with Lipofectamine™ 2000 (Invitrogen GmbH, Karlsruhe, Germany), with the plasmid diluted in Opti-MEM to a final volume of 12.5 μl per well, prepared as a mastermix for the whole plate.

4 h after plasmid transfection, growth medium was removed from cells and replaced by 100 μl/well of fresh medium. siRNA transfections were performed using Lipofectamine™ 2000 (Invitrogen GmbH, Karlsruhe, Germany) as described by the manufacturer. Cells were incubated for 24 h at 37° C. and 5% CO₂ in a humidified incubator (Heraeus GmbH, Hanau, Germany). For the primary screen, all siRNAs were screened at a final concentration of 30 nM. Selected sequences were rescreened at a siRNA concentration of 300 pM. Each siRNA was tested in quadruplicate for each concentration.

Cells were lysed by removing growth medium and application of 150 μl of a 1:1 mixture consisting of medium and substrate from the Dual-Glo Luciferase Assay System (Promega, Mannheim, Germany). The luciferase assay was performed according to the manufacturer's protocol for Dual-Glo Luciferase assay and luminescence was measured in a Victor-Light 1420 Luminescence Counter (Perkin Elmer, Rodgau-Juigesheim, Germany). Values obtained with Renilla luciferase were normalized to the respective values obtained with Firefly luciferase in order to correct for transfection efficacy. Renilla/Firefly luciferase activities obtained after transfection with siRNAs directed against a JCV gene were normalized to Renilla/Firefly luciferase activities obtained after transfection of an unrelated control siRNA set to 100%. Tables 1a and b provides the results where the siRNAs, the sequences of which are given in Tables 1a and b, were tested at a single dose of 30 nM. The percentage inhibition±standard deviation, compared to the unrelated control siRNA, is indicated in the column ‘Remaining luciferase activity (% of control)’. A number of JCV siRNAs at 30 nM were effective at reducing levels of the targeted mRNA by more than 70% in Cos-7 cells (i.e. remaining luciferase activity was less than 30%).

Selected JCV siRNAs from the single dose screen were further characterized by dose response curves. Transfections of JCV siRNAs for generation of dose response curves were performed with the following siRNA concentrations according to the above protocol:

-   -   from 33 nM in 3-fold dilutions down to 0.005 nM (for fragment         L1)     -   from 24 nM in 4-fold dilutions down to 0.001 nM (for fragment E         and fragments LA23 1-700 and LA23 701-1438).

IC50 values were determined by parameterized curve fitting using the program XLfit (IDBS, Guildford, Great Britain). Table 2 provides the results from two independent experiments for 32 selected JCV siRNAs. The mean IC50 from these two independent experiments is shown. Several JCV siRNAs (AD-12622, AD-12677, AD-12709, AD-12710, AD-12722, AD-12724, AD-12728, AD-12763, AD-12767, AD-12768, AD-12769, AD-12771, AD-12774, AD-12775, AD-12777, AD-12781, AD-12784, AD-12795, AD-12813, AD-12821, AD-12823, AD-12824, AD-12825, AD-12827, AD-12829, AD-12842) were particularly potent in this experimental paradigm, and exhibited IC50 values between 70 μM and 1 nM.

TABLE 2 IC50s Mean IC50 Duplex name [nM] AD-12599 2.37 AD-12622 0.57 AD-12666 3.7 AD-12677 0.49 AD-12709 0.19 AD-12710 0.47 AD-12712 2.33 AD-12722 0.12 AD-12724 0.26 AD-12728 0.8 AD-12761 1.2 AD-12763 0.95 AD-12767 0.09 AD-12768 0.19 AD-12769 0.35 AD-12771 0.35 AD-12774 0.13 AD-12775 0.18 AD-12777 0.17 AD-12778 12.65 AD-12781 0.18 AD-12784 0.44 AD-12795 0.65 AD-12813 0.2 AD-12818 1.88 AD-12821 0.07 AD-12823 0.46 AD-12824 0.25 AD-12825 0.52 AD-12827 0.15 AD-12829 0.14 AD-12842 0.44 Screen of JCV siRNAs Against Live JC Virus in SVG-A Cells

Cells and Virus

SVG-A cells (human fetal glial cells transformed by SV40 T antigen) obtained from Walter Atwood at Brown University were cultured in Eagle's Minimum Essential Media (ATCC, Manassas, Va.) supplemented to contain 10% fetal bovine serum (FBS) (Omega Scientific, Tarzana, Calif.), Penicillin 100 U/ml, Streptomycin 100 ug/ml (Invitrogen, Carlsbad Calif.) at 37° C. in an atmosphere with 5% CO₂ in a humidified incubator (Heraeus HERAcell, Thermo Electron Corporation, Ashville, N.C.). The Mad-1-SVEΔ strain of JCV obtained from Walter Atwood at Brown University was used in all experiments; viral stocks were prepared using SVG-A cells according to standard published methods (Liu and Atwood, Propagation and assay of the JC Virus, Methods Mol. Biol. 2001; 165:9-17).

Prophylaxis Assay

SVG-A cells were seeded on glass coverslips in 6-well dishes 24 hours prior to transfection in the media described above minus antibiotics. Cells were transfected with the indicated concentration of siRNA (10 nM, 50 nM, or 100 nM) using Lipofectamine™ 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.). Twenty-four hours post-transfection cells were washed with media containing 2% FBS and then infected with a 1:25 dilution of JCV virus stock (Mad-1-SVEΔ strain) diluted in 2% FBS media. Cells were rocked every 15 minutes by hand several times to get equal virus binding across the entire coverslip for one hour and then additional 10% FBS media was added and the infection was allowed to proceed for 72 hours. Seventy two hours post-infection, cells were fixed in acetone and stained for late viral protein (VP1) by standard immunofluoresence methods using hybridoma supernatant PAB597 recognizing JCV VP1 (obtained from Walter Atwood at Brown University) with goat anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen, Carlsbad, Calif.). Infected cells were scored by counting VP1-immunoreactive cells using a fluorescence microscope (Zeiss, Imager.Z1, Thornwood, N.J.) and data were expressed as the percentage of infected cells counted for the control coverslips transfected with Luciferase siRNA. Table 3 shows the results of the prophylaxis assays at different siRNA concentrations (10 nM, 50 nM or 100 nM). The VP1 siRNAs were the most potent as a group, followed by the T antigen siRNAs, with the VP2/3 siRNAs being the least potent. The VP1 siRNAs most effective in reducing virus were consistently AD-12622, AD-12728, AD-12795, and AD-12842. The most potent T antigen siRNA was AD-12813.

TABLE 3 Prophylaxis Assay Remaining Virus Duplex Targeted JCV (% of Luciferase Control) Number Transcript 50 nM 10 nM 100 nM AD-12599 VP1 79.9 ND ND AD-12709 VP1 46.0 ND ND AD-12710 VP1 25.9 ND ND AD-12784 VP1 30.9 ND ND AD-12712 VP1 29.7 ND ND AD-12724 VP1 30.5 38.9 25.8 AD-12622 VP1 22.9 28.2  9.1 AD-12728 VP1 21.1 22.2 ND AD-12795 VP1 13.6 16.9  8.5 AD-12842 VP1 16.0 23.4 12.7 AD-12761 VP1 26.4 52.3 ND AD-12818 VP1 24.0 50.2 28.0 AD-12666 VP1 54.1 ND ND AD-12763 VP1 39.5 ND ND AD-12722 T Antigen 43.6 82.1 ND AD-12813 T Antigen 21.5 48.8 19.4 AD-12767 T Antigen 37.6 52.2 30.9 AD-12821 T Antigen 33.0 51.2 30.8 AD-12774 T Antigen 74.0 89.2 ND AD-12827 T Antigen 77.0 92.0 ND AD-12775 T Antigen 81.6 95.4 ND AD-12777 T Antigen 73.3 93.9 ND AD-12829 T Antigen 78.6 93.6 ND AD-12781 T Antigen 38.8 62.6 34.4 AD-12768 VP2/3 73.9 92.4 ND AD-12771 VP2/3 51.6 83.6 ND AD-12824 VP2/3 42.1 79.0 43.7 AD-12769 VP2/3 35.2 78.0 39.7 AD-12823 VP2/3 38.1 78.1 42.0 AD-12677 VP2/3 99.1 102.1  ND AD-12825 VP2/3 100.8 99.1 ND ND indicates no data.

Post-Infection Treatment Assay

SVG-A cells were seeded on glass coverslips in 6-well dishes 24 hours prior to infection in 10% FBS media. Cells were washed with media containing 2% FBS and then infected with a 1:25 dilution of JCV virus stock diluted in 2% FBS media. Cells were rocked by hand approximately 8-10 times to get equal virus binding across the entire coverslip every 15 minutes for one hour and then additional 10% FBS media was added. Twenty-four and forty-eight hours postinfection, cells were washed with 10% FBS media containing no antibiotics and then transfected with 50 nM of the indicated siRNA using Lipofectamine™ 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.). Seventy-two hours postinfection, cells were fixed in acetone and stained for late viral protein (VP1) by standard immunofluoresence methods using hybridoma supernatant PAB597 recognizing JCV VP1 (obtained from Walter Atwood at Brown University) with goat anti-mouse Alexa Fluor 488 secondary antibody (Molecular Probes, Eugene, Oreg.). Infected cells were scored by counting VP1-immunoreactive cells using a fluorescence microscope (Zeiss, Imager.Z 1, Thornwood, N.J.) and data were expressed as the percentage of infected cells counted for control coverslips transfected with Luciferase siRNA. Table 4 shows the results of the post-infection treatment experiments. All of the siRNAs tested in the treatment assay showed significant antiviral activity against JCV, such that the remaining virus was significantly less than that in the luciferase siRNA control.

TABLE 4 Treatment Assay Targeted JCV Remaining Virus (% of Duplex Number Transcript Luciferase Control) AD-12724 VP1 38.9 AD-12622 VP1 28.2 AD-12795 VP1 16.9 AD-12842 VP1 23.4 AD-12818 VP1 ND AD-12813 T Antigen 48 AD-12767 T Antigen 56.9 AD-12821 T Antigen 75.8 AD-12781 T Antigen 75.8 AD-12824 VP2/3 60.4 AD-12769 VP2/3 70.7 AD-12823 VP2/3 72.4 ND indicates no data. Prophylaxis Administration of JCV siRNAs Inhibits the Production of Active Progeny JC Virus

SVG-A cells were seeded in 6-well dishes 24 hours prior to transfection in the media described above minus antibiotics. Cells were transfected with 10 nM of the indicated siRNA using Lipofectamine™ 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif.). Twenty-four hours post-transfection cells were washed with media containing 2% FBS and then infected with a 1:25 dilution of JCV virus stock (Mad-1-SVEΔ strain) diluted in 2% FBS media. Cells were rocked every 15 minutes by hand several times to get equal virus binding across the entire coverslip for one hour and then additional 10% FBS media was added and the infection was allowed to proceed for 6 days. Six days post-infection, progeny virus was collected either by removal of overlay media from infected cells or by scraping cells and performing virus preparations. The virus preparations consisted of scraping cells into the supernatant media, vortexing, freeze-thawing the re-suspended cells 2 times with vortexing in between, then spinning down the cell debris and taking the supernatant. Fresh SVG-A cells seeded on glass coverslips were infected secondarily with virus collected by either method using the same procedure done with the initial infection to determine the amount of infectious virus produced by cells transfected with the various siRNAs. At 72 hours post-infection of coverslips, cells were fixed in acetone and stained for late viral protein (VP1) by standard immunofluoresence methods using hybridoma supernatant PAB597 recognizing JCV VP1 (obtained from Walter Atwood at Brown University) with goat anti-mouse Alexa Fluor 488 secondary antibody (Invitrogen, Carlsbad, Calif.). Infected cells were scored by counting VP1-immunoreactive cells using a fluorescence microscope (Zeiss, Imager.Z1, Thornwood, N.J.) and data were expressed as the percentage of infected cells counted for the control coverslips transfected with Luciferase siRNA. Table 5 shows the results for selected siRNAs, demonstrating the ability of prophylaxis siRNA treatment to inhibit active progeny virus production by either method of virus collection. Transfection with siRNAs targeting VP1 (AD-12622 and AD-12842) had the greatest effect on inhibiting the production of active progeny virus regardless of whether virus was collected from media or from infected cell preparations. The T antigen siRNA AD-12813 had the next strongest inhibitory effect, whereas the VP2/3 siRNAs AD-12824 and AD-12769 still showed some albeit a lesser ability to inhibit active progeny JCV production.

TABLE 5 Prophylaxis administration of JCV siRNAs inhibits the production of active progeny JC virus capable of secondary infection Remaining Virus (% of Luciferase Control) Targeted Virus Duplex Name Transcript Media Preparation AD-12622 VP1 30.8 24.9 AD-12842 VP1 33.3 26.9 AD-12813 T Antigen 57.8 38.7 AD-12824 VP2/3 83.6 57.6 AD-12769 VP2/3 79.1 52.2 Stability in Cerebrospinal Fluid (CSF) of Selected siRNAs Targeting JCV

Eleven selected JCV siRNAs were tested for stability at 5 uM over 48 h at 37° C. in human CSF, as well as in PBS for comparison. 30 μl of human cerebrospinal fluid (CSF) was mixed with 3 μl of 50 μM duplex (siRNA) solution (150 pmole/well) in a 96-well plate, sealed to avoid evaporation and incubated for the indicated time at 37° C. Incubation of the siRNA in 30 ul PBS for 48 h served as a control for non-specific degradation. Reactions were stopped by the addition of 4 ul proteinase K (20 mg/ml) and 25 ul of proteinase K buffer, and an incubation for 20′ at 42° C. Samples were then spin filtered through a 0.2 μm 96 well filter plate at 3000 rpm for 20′. Incubation wells were washed with 50 ul Millipore water twice and the combined washing solutions were spin filtered also.

Samples were analyzed by ion exchange HPLC under denaturing conditions. Samples were transferred to single autosampler vials. IEX-HPLC analysis was performed under the following conditions: Dionex DNAPac PA200 (4×250 mm analytical column), temperature of 45° C. (denaturing conditions by pH=11), flow rate of 1 ml/min, injection volume of 50 ul, and detection wavelength of 260 nm with 1 nm bandwidth (reference wavelength 600 nm). In addition, the gradient conditions were as follows with HPLC Eluent A: 20 mM Na₃PO₄ in 10% ACN; pH=11 and HPLC Eluent B: 1 M NaBr in HPLC Eluent A:

Time % A % B 0.00 min 75 25 1.00 min 75 25 19.0 min 38 62 19.5 min 0 100 21.5 min 0 100 22.0 min 75 25 24.0 min 75 25

Under the above denaturing IEX-HPLC conditions, the duplexes eluted as two separated single strands. All chromatograms were integrated automatically by the Dionex Chromeleon 6.60 HPLC software, and were adjusted manually as necessary. The area under the peak for each strand was calculated and the %-values for each intact full length product (FLP) for each time points were calculated by the following equation:

%-FLP_((s/as; t=x))=(PeakArea_((s/as);t=x)/PeakArea_((s/as);t=0min))*100%

All values were normalized to FLP at t=0 min. Table 6 provides the results after 48 hours of incubation in human CSF at 37° C. At least 75% of both antisense and sense strands of ten JCV siRNAs (AD-12622, AD-12724, AD-12767, AD-12769, AD-12795, AD-12813, AD-12818, AD-12823, AD-12824, AD-12842) were recovered, demonstrating that these siRNAs are highly stable in human CSF at 37° C. For AD-12821, 59% of the antisense and 97% of the sense strand was recovered after 48 h of incubation in human CSF at 37° C., showing that this siRNA has a half-life of greater than 48 h in human CSF at 37° C.

TABLE 6 Stability in human CSF % full length material after Duplex 48 hours name antisense sense AD-12622 93 105 AD-12724 90 106 AD-12767 85 104 AD-12769 100 104 AD-12795 86 109 AD-12813 94 98 AD-12818 75 99 AD-12821 59 97 AD-12823 98 98 AD-12824 84 98 AD-12842 87 102

dsRNA Expression Vectors

In another aspect of the invention, JC virus specific dsRNA molecules that modulate JC virus genome expression activity are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.

The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or Ul snRNA promoter) or generally RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g. the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single JC virus genome or multiple JC virus genomes over a period of a week or more are also contemplated by the invention. Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection. can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection. of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

The JC virus specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

JCV Gene Walk siRNAs targeting >95% of all strains (>=369 out of 388) Human specific pan-JCV: 208 siRNAs all siRNAs double overhang design, dTdT, no modifications Residual luciferase activity (relative to sense strand antisense strand control SEQ SEQ siRNA position in duplex ID ID treated consensus name NO: sequence (5′-3′) NO: sequence (5′-3′) cells) 1533-1551 AD-14742 515 CUUAUAAGAGGAGGAGUAGTT 516 CUACUCCUCCUCUUAUAAGTT 40.85 1703-1721 AD-14743 517 CAUGCUUCCUUGUUACAGUTT 518 ACUGUAACAAGGAAGCAUGTT 20.92 1439-1457 AD-14744 519 UACGGGACUGUAACACCUGTT 520 CAGGUGUUACAGUCCCGUATT 62.20 1705-1723 AD-14745 521 UGCUUCCUUGUUACAGUGUTT 522 ACACUGUAACAAGGAAGCATT 43.97 2064-2082 AD-14746 523 CCUGUUGAAUGUUGGGUUCTT 524 GAACCCAACAUUCAACAGGTT 24.52 2067-2085 AD-14747 525 GUUGAAUGUUGGGUUCCUGTT 526 CAGGAACCCAACAUUCAACTT 32.67 2071-2089 AD-14748 527 AAUGUUGGGUUCCUGAUCCTT 528 GGAUCAGGAACCCAACAUUTT 93.99 2121-2139 AD-14749 529 ACACUAACAGGAGGAGAAATT 530 UUUCUCCUCCUGUUAGUGUTT 55.16 1535-1553 AD-14750 531 UAUAAGAGGAGGAGUAGAATT 532 UUCUACUCCUCCUCUUAUATT 30.86 1536-1554 AD-14751 533 AUAAGAGGAGGAGUAGAAGTT 534 CUUCUACUCCUCCUCUUAUTT 54.44 1445-1463 AD-14752 535 ACUGUAACACCUGCUCUUGTT 536 CAAGAGCAGGUGUUACAGUTT 53.88 1700-1718 AD-14753 537 GGACAUGCUUCCUUGUUACTT 538 GUAACAAGGAAGCAUGUCCTT 35.24 1702-1720 AD-14754 539 ACAUGCUUCCUUGUUACAGTT 540 CUGUAACAAGGAAGCAUGUTT 70.39 1704-1722 AD-14755 541 AUGCUUCCUUGUUACAGUGTT 542 CACUGUAACAAGGAAGCAUTT 41.80 2065-2083 AD-14756 543 CUGUUGAAUGUUGGGUUCCTT 544 GGAACCCAACAUUCAACAGTT 56.69 2070-2088 AD-14757 545 GAAUGUUGGGUUCCUGAUCTT 546 GAUCAGGAACCCAACAUUCTT 39.16 1441-1459 AD-14758 547 CGGGACUGUAACACCUGCUTT 548 AGCAGGUGUUACAGUCCCGTT 39.79 1443-1461 AD-14759 549 GGACUGUAACACCUGCUCUTT 550 AGAGCAGGUGUUACAGUCCTT 30.62 1444-1462 AD-14760 551 GACUGUAACACCUGCUCUUTT 552 AAGAGCAGGUGUUACAGUCTT 28.14 1609-1627 AD-14761 553 CUCCAGAAAUGGGUGACCCTT 554 GGGUCACCCAUUUCUGGAGTT 67.42 1537-1555 AD-14762 555 UAAGAGGAGGAGUAGAAGUTT 556 ACUUCUACUCCUCCUCUUATT 36.10 629-647 AD-14763 557 GAGGCUGCUGCUACUAUAGTT 558 CUAUAGUAGCAGCAGCCUCTT 49.39 656-674 AD-14764 559 AUUGCAUCCCUUGCUACUGTT 560 CAGUAGCAAGGGAUGCAAUTT 74.04 658-676 AD-14765 561 UGCAUCCCUUGCUACUGUATT 562 UACAGUAGCAAGGGAUGCATT 50.84 517-535 AD-14766 563 UUGUGUUUUCAGGUUCAUGTT 564 CAUGAACCUGAAAACACAATT 72.59 559-577 AD-14767 565 GGACCUAGUUGCUACUGUUTT 566 AACAGUAGCAACUAGGUCCTT 34.82 591-609 AD-14768 567 CUGCCACAGGAUUUUCAGUTT 568 ACUGAAAAUCCUGUGGCAGTT 48.68 638-656 AD-14769 569 GCUACUAUAGAAGUUGAAATT 570 UUUCAACUUCUAUAGUAGCTT 39.07 655-673 AD-14770 571 AAUUGCAUCCCUUGCUACUTT 572 AGUAGCAAGGGAUGCAAUUTT 45.59 561-579 AD-14771 573 ACCUAGUUGCUACUGUUUCTT 574 GAAACAGUAGCAACUAGGUTT 45.57 639-657 AD-14772 575 CUACUAUAGAAGUUGAAAUTT 576 AUUUCAACUUCUAUAGUAGTT 33.12 715-733 AD-14773 577 AGGCCUUACUCCUGAAACATT 578 UGUUUCAGGAGUAAGGCCUTT 37.38 716-734 AD-14774 579 GGCCUUACUCCUGAAACAUTT 580 AUGUUUCAGGAGUAAGGCCTT 42.38 326-344 AD-14775 581 GUAAAACCUGGAGUGGAACTT 582 GUUCCACUCCAGGUUUUACTT 46.59 518-536 AD-14776 583 UGUGUUUUCAGGUUCAUGGTT 584 CCAUGAACCUGAAAACACATT 71.28 520-538 AD-14777 585 UGUUUUCAGGUUCAUGGGUTT 586 ACCCAUGAACCUGAAAACATT 64.55 661-679 AD-14778 587 AUCCCUUGCUACUGUAGAGTT 588 CUCUACAGUAGCAAGGGAUTT 60.45 560-578 AD-14779 589 GACCUAGUUGCUACUGUUUTT 590 AAACAGUAGCAACUAGGUCTT 32.46 681-699 AD-14780 591 GGAUUACAAGUACCUCUGATT 592 UCAGAGGUACUUGUAAUCCTT 22.96 714-732 AD-14781 593 UAGGCCUUACUCCUGAAACTT 594 GUUUCAGGAGUAAGGCCUATT 56.99 377-395 AD-14782 595 UGUUAGAAUUUUUGCUGGATT 596 UCCAGCAAAAAUUCUAACATT 29.90 589-607 AD-14783 597 UGCUGCCACAGGAUUUUCATT 598 UGAAAAUCCUGUGGCAGCATT 42.63 594-612 AD-14784 599 CCACAGGAUUUUCAGUAGCTT 600 GCUACUGAAAAUCCUGUGGTT 67.06 648-666 AD-14785 601 AAGUUGAAAUUGCAUCCCUTT 602 AGGGAUGCAAUUUCAACUUTT 48.90 649-667 AD-14786 603 AGUUGAAAUUGCAUCCCUUTT 604 AAGGGAUGCAAUUUCAACUTT 27.74 587-605 AD-14787 605 GCUGCUGCCACAGGAUUUUTT 606 AAAAUCCUGUGGCAGCAGCTT 38.77 325-343 AD-14788 607 AGUAAAACCUGGAGUGGAATT 608 UUCCACUCCAGGUUUUACUTT 32.84 515-533 AD-14789 609 UUUUGUGUUUUCAGGUUCATT 610 UGAACCUGAAAACACAAAATT 46.96 516-534 AD-14790 611 UUUGUGUUUUCAGGUUCAUTT 612 AUGAACCUGAAAACACAAATT 43.61 519-537 AD-14791 613 GUGUUUUCAGGUUCAUGGGTT 614 CCCAUGAACCUGAAAACACTT 35.55 521-539 AD-14792 615 GUUUUCAGGUUCAUGGGUGTT 616 CACCCAUGAACCUGAAAACTT 38.22 522-540 AD-14793 617 UUUUCAGGUUCAUGGGUGCTT 618 GCACCCAUGAACCUGAAAATT 90.85 523-541 AD-14794 619 UUUCAGGUUCAUGGGUGCCTT 620 GGCACCCAUGAACCUGAAATT 83.37 616-634 AD-14795 621 UUGCAUCCCUUGCUACUGUTT 622 AGCCUCUCCAGCAGCAAUUTT 55.06 657-675 AD-14796 623 UUGCAUCCCUUGCUACUGUTT 624 ACAGUAGCAAGGGAUGCAATT 30.98 761-779 AD-14797 625 GCUGUAGCUGGGUUUGCUGTT 626 CAGCAAACCCAGCUACAGCTT 28.95 645-663 AD-14798 627 UAGAAGUUGAAAUUGCAUCTT 628 GAUGCAAUUUCAACUUCUATT 67.39 647-665 AD-14799 629 GAAGUUGAAAUUGCAUCCCTT 630 GGGAUGCAAUUUCAACUUCTT 66.83 660-678 AD-14800 631 CAUCCCUUGCUACUGUAGATT 632 UCUACAGUAGCAAGGGAUGTT 33.26 324-342 AD-14801 633 UAGUAAAACCUGGAGUGGATT 634 UCCACUCCAGGUUUUACUATT 39.15 372-390 AD-14802 635 UUUUUUGUUAGAAUUUUUGTT 636 CAAAAAUUCUAACAAAAAATT 91.20 640-658 AD-14803 637 UACUAUAGAAGUUGAAAUUTT 638 AAUUUCAACUUCUAUAGUATT 34.15 562-580 AD-14804 639 CCUAGUUGCUACUGUUUCUTT 640 AGAAACAGUAGCAACUAGGTT 30.08 563-581 AD-14805 641 CUAGUUGCUACUGUUUCUGTT 642 CAGAAACAGUAGCAACUAGTT 32.44 566-584 AD-14806 643 GUUGCUACUGUUUCUGAGGTT 644 CCUCAGAAACAGUAGCAACTT 35.62 625-643 AD-14807 645 UGGAGAGGCUGCUGCUACUTT 646 AGUAGCAGCAGCCUCUCCATT 28.27 627-645 AD-14808 647 GAGAGGCUGCUGCUACUAUTT 648 AUAGUAGCAGCAGCCUCUCTT 30.29 628-646 AD-14809 649 AGAGGCUGCUGCUACUAUATT 650 UAUAGUAGCAGCAGCCUCUTT 31.59 632-650 AD-14810 651 GCUGCUGCUACUAUAGAAGTT 652 CUUCUAUAGUAGCAGCAGCTT 30.11 513-531 AD-14811 653 UUUUUUGUGUUUUCAGGUUTT 654 AACCUGAAAACACAAAAAATT 55.27 641-659 AD-14812 655 ACUAUAGAAGUUGAAAUUGTT 656 CAAUUUCAACUUCUAUAGUTT 45.27 323-341 AD-14813 657 UUAGUAAAACCUGGAGUGGTT 658 CCACUCCAGGUUUUACUAATT 77.97 717-735 AD-14814 659 GCCUUACUCCUGAAACAUATT 660 UAUGUUUCAGGAGUAAGGCTT 29.54 646-664 AD-14815 661 AGAAGUUGAAAUUGCAUCCTT 662 GGAUGCAAUUUCAACUUCUTT 65.04 592-610 AD-14816 663 UGCCACAGGAUUUUCAGUATT 664 UACUGAAAAUCCUGUGGCATT 64.03 590-608 AD-14817 665 GCUGCCACAGGAUUUUCAGTT 666 CUGAAAAUCCUGUGGCAGCTT 37.83 526-544 AD-14818 667 CAGGUUCAUGGGUGCCGCATT 668 UGCGGCACCCAUGAACCUGTT 28.88 615-633 AD-14819 669 AAAUUGCUGCUGGAGAGGCTT 670 GCCUCUCCAGCAGCAAUUUTT 92.90 617-635 AD-14820 671 AUUGCUGCUGGAGAGGCUGTT 672 CAGCCUCUCCAGCAGCAAUTT 75.41 652-670 AD-14821 673 UGAAAUUGCAUCCCUUGCUTT 674 AGCAAGGGAUGCAAUUUCATT 73.08 374-392 AD-14822 675 UUUUGUUAGAAUUUUUGCUTT 676 AGCAAAAAUUCUAACAAAATT 86.39 375-393 AD-14823 677 UUUGUUAGAAUUUUUGCUGTT 678 CAGCAAAAAUUCUAACAAATT 96.50 631-649 AD-14824 679 GGCUGCUGCUACUAUAGAATT 680 UUCUAUAGUAGCAGCAGCCTT 32.62 376-394 AD-14825 681 UUGUUAGAAUUUUUGCUGGTT 682 CCAGCAAAAAUUCUAACAATT 102.71 512-530 AD-14826 683 UUUUUUUGUGUUUUCAGGUTT 684 ACCUGAAAACACAAAAAAATT 92.45 1127-1145 AD-14827 685 GAAACUACUUGGGCAAUAGTT 686 CUAUUGCCCAAGUAGUUUCTT 63.46 1410-1428 AD-14828 687 AAUGGAUGUUGCCUUUACUTT 688 AGUAAAGGCAACAUCCAUUTT 45.99 1406-1424 AD-14829 689 CCUCAAUGGAUGUUGCCUUTT 690 AAGGCAACAUCCAUUGAGGTT 40.54 1418-1436 AD-14830 691 UUGCCUUUACUUUUAGGGUTT 692 ACCCUAAAAGUAAAGGCAATT 117.10 1126-1144 AD-14831 693 AGAAACUACUUGGGCAAUATT 694 UAUUGCCCAAGUAGUUUCUTT 54.78 1125-1143 AD-14832 695 AAGAAACUACUUGGGCAAUTT 696 AUUGCCCAAGUAGUUUCUUTT 67.07 1419-1437 AD-14833 697 UGCCUUUACUUUUAGGGUUTT 698 AACCCUAAAAGUAAAGGCATT 71.52 1420-1438 AD-14834 699 GCCUUUACUUUUAGGGUUGTT 700 CAACCCUAAAAGUAAAGGCTT 58.05 1422-1440 AD-14835 701 CUUUACUUUUAGGGUUGUATT 702 UACAACCCUAAAAGUAAAGTT 93.36 1423-1441 AD-14836 703 UUUACUUUUAGGGUUGUACTT 704 GUACAACCCUAAAAGUAAATT 108.84 1425-1443 AD-14837 705 UACUUUUAGGGUUGUACGGTT 706 CCGUACAACCCUAAAAGUATT 106.68 1123-1141 AD-14838 707 GGAAGAAACUACUUGGGCATT 708 UGCCCAAGUAGUUUCUUCCTT 37.06 1409-1427 AD-14839 709 CAAUGGAUGUUGCCUUUACTT 710 GUAAAGGCAACAUCCAUUGTT 36.03 1413-1431 AD-14840 711 GGAUGUUGCCUUUACUUUUTT 712 AAAAGUAAAGGCAACAUCCTT 38.51 1416-1434 AD-14841 713 UGUUGCCUUUACUUUUAGGTT 714 CCUAAAAGUAAAGGCAACATT 110.86 1414-1432 AD-14842 715 GAUGUUGCCUUUACUUUUATT 716 UAAAAGUAAAGGCAACAUCTT 34.83 911-929 AD-14843 717 CCAGAAGACUACUAUGAUATT 718 UAUCAUAGUAGUCUUCUGGTT 23.75 910-928 AD-14844 719 UCCAGAAGACUACUAUGAUTT 720 AUCAUAGUAGUCUUCUGGATT 27.47 1120-1138 AD-14845 721 UUUGGAAGAAACUACUUGGTT 722 CCAAGUAGUUUCUUCCAAATT 93.12 1404-1422 AD-14846 723 CUCCUCAAUGGAUGUUGCCTT 724 GGCAACAUCCAUUGAGGAGTT 81.72 1337-1355 AD-14847 725 CCAAAUGUGCAAUCUGGUGTT 726 CACCAGAUUGCACAUUUGGTT 77.89 1338-1356 AD-14848 727 CAAAUGUGCAAUCUGGUGATT 728 UCACCAGAUUGCACAUUUGTT 44.40 1397-1415 AD-14849 729 AGAUCUGCUCCUCAAUGGATT 730 UCCAUUGAGGAGCAGAUCUTT 46.41 1407-1425 AD-14850 731 CUCAAUGGAUGUUGCCUUUTT 732 AAAGGCAACAUCCAUUGAGTT 35.52 4157-4175 AD-14851 733 GCUCAAAUUUUAUAUAAGATT 734 UCUUAUAUAAAAUUUGAGCTT 36.07 4795-4813 AD-14852 735 AGCCUGAUUUUGGUACAUGTT 736 CAUGUACCAAAAUCAGGCUTT 67.98 4156-4174 AD-14853 737 CUCAAAUUUUAUAUAAGAATT 738 UUCUUAUAUAAAAUUUGAGTT 69.44 5002-5020 AD-14854 739 ACAAAGUGCUGAAUAGGGATT 740 UCCCUAUUCAGCACUUUGUTT 29.12 4792-4810 AD-14855 741 CUGAUUUUGGUACAUGGAATT 742 UUCCAUGUACCAAAAUCAGTT 36.04 4790-4808 AD-14856 743 GAUUUUGGUACAUGGAAUATT 744 UAUUCCAUGUACCAAAAUCTT 33.61 4801-4819 AD-14857 745 CUCAUCAGCCUGAUUUUGGTT 746 CCAAAAUCAGGCUGAUGAGTT 50.76 4622-4640 AD-14858 747 AGCCCACUUGUGUGGAUAGTT 748 CUAUCCACACAAGUGGGCUTT 53.60 4997-5015 AD-14859 749 GUGCUGAAUAGGGAGGAAUTT 750 AUUCCUCCCUAUUCAGCACTT 39.07 5094-5112 AD-14860 751 AGUAAGGGCGUGGAGGCUUTT 752 AAGCCUCCACGCCCUUACUTT 62.78 4564-4582 AD-14861 753 GUGACUUAACCCAAGAAGCTT 754 GCUUCUUGGGUUAAGUCACTT 87.47 5095-5113 AD-14862 755 UAGUAAGGGCGUGGAGGCUTT 756 AGCCUCCACGCCCUUACUATT 79.95 4800-4818 AD-14863 757 UCAUCAGCCUGAUUUUGGUTT 758 ACCAAAAUCAGGCUGAUGATT 30.46 4265-4283 AD-14864 759 GUAGAAGACCCUAAAGACUTT 760 AGUCUUUAGGGUCUUCUACTT 33.18 4267-4285 AD-14865 761 AGGUAGAAGACCCUAAAGATT 762 UCUUUAGGGUCUUCUACCUTT 26.25 4270-4288 AD-14866 763 AAAAGGUAGAAGACCCUAATT 764 UUAGGGUCUUCUACCUUUUTT 36.73 4269-4287 AD-14867 765 AAAGGUAGAAGACCCUAAATT 766 UUUAGGGUCUUCUACCUUUTT 33.16 2874-2892 AD-14868 767 GAUUGUGCAGUGGAAAGAATT 768 UUCUUUCCACUGCACAAUCTT 29.91 2875-2893 AD-14869 769 GGAUUGUGCAGUGGAAAGATT 770 UCUUUCCACUGCACAAUCCTT 28.24 3950-3968 AD-14870 771 UGUAGACAGCCAUAUGCAGTT 772 CUGCAUAUGGCUGUCUACATT 50.37 3896-3914 AD-14871 773 CAUGACUUUAACCCAGAAGTT 774 CUUCUGGGUUAAAGUCAUGTT 39.37 4990-5008 AD-14872 775 AUAGGGAGGAAUCCAUGGATT 776 UCCAUGGAUUCCUCCCUAUTT 34.71 4994-5012 AD-14873 777 CUGAAUAGGGAGGAAUCCATT 778 CCUCCCUAUUCAGCACUUUTT 32.14 5000-5018 AD-14874 779 AAAGUGCUGAAUAGGGAGGTT 780 CCUCCCUAUUCAGCACUUUTT 101.77 4563-4581 AD-14875 781 UGACUUAACCCAAGAAGCUTT 782 AGCUUCUUGGGUUAAGUCATT 80.81 3895-3913 AD-14876 783 AUGACUUUAACCCAGAAGATT 784 UCUUCUGGGUUAAAGUCAUTT 30.74 4262-4280 AD-14877 785 GAAGACCCUAAAGACUUUCTT 786 GAAAGUCUUUAGGGUCUUCTT 57.38 4162-4180 AD-14878 787 AAAAAGCUCAAAUUUUAUATT 788 UAUAAAAUUUGAGCUUUUUTT 70.23 4798-4816 AD-14879 789 AUCAGCCUGAUUUUGGUACTT 790 GUACCAAAAUCAGGCUGAUTT 79.03 4799-4817 AD-14880 791 CAUCAGCCUGAUUUUGGUATT 792 UACCAAAAUCAGGCUGAUGTT 21.65 5006-5024 AD-14881 793 AUGGACAAAGUGCUGAAUATT 794 UAUUCAGCACUUUGUCCAUTT 27.66 4264-4282 AD-14882 795 UAGAAGACCCUAAAGACUUTT 796 AAGUCUUUAGGGUCUUCUATT 34.01 4268-4286 AD-14883 797 AAGGUAGAAGACCCUAAAGTT 798 CUUUAGGGUCUUCUACCUUTT 40.62 4623-4641 AD-14884 799 CAGCCCACUUGUGUGGAUATT 800 UAUCCACACAAGUGGGCUGTT 35.73 4788-4806 AD-14885 801 UUUUGGUACAUGGAAUAGUTT 802 ACUAUUCCAUGUACCAAAATT 47.40 4993-5011 AD-14886 803 UGAAUAGGGAGGAAUCCAUTT 804 AUGGAUUCCUCCCUAUUCATT 37.23 4995-5013 AD-14887 805 GCUGAAUAGGGAGGAAUCCTT 806 GGAUUCCUCCCUAUUCAGCTT 42.94 4996-5014 AD-14888 807 UGCUGAAUAGGGAGGAAUCTT 808 GAUUCCUCCCUAUUCAGCATT 32.58 3952-3970 AD-14889 809 UGUGUAGACAGCCAUAUGCTT 810 GCAUAUGGCUGUCUACACATT 83.09 4595-4613 AD-14890 811 UGCUUUGAUUGCUUCAGACTT 812 GUCUGAAGCAAUCAAAGCATT 59.49 4596-4614 AD-14891 813 UUGCUUUGAUUGCUUCAGATT 814 UCUGAAGCAAUCAAAGCAATT 21.93 4597-4615 AD-14892 815 AUUGCUUUGAUUGCUUCAGTT 816 CUGAAGCAAUCAAAGCAAUTT 72.69 4599-4617 AD-14893 817 CUAUUGCUUUGAUUGCUUCTT 818 GAAGCAAUCAAAGCAAUAGTT 24.43 4726-4744 AD-14894 819 AUUGCAAGGAAUGGCCUAATT 820 UUAGGCCAUUCCUUGCAAUTT 33.84 4753-4771 AD-14895 821 AUUUUCCUCCUAAUUCUGATT 822 UCAGAAUUAGGAGGAAAAUTT 21.68 4802-4820 AD-14896 823 GCUCAUCAGCCUGAUUUUGTT 824 CAAAAUCAGGCUGAUGAGCTT 26.99 4803-4821 AD-14897 825 UGCUCAUCAGCCUGAUUUUTT 826 AAAAUCAGGCUGAUGAGCATT 29.04 4806-4824 AD-14898 827 AGUUGCUCAUCAGCCUGAUTT 828 AUCAGGCUGAUGAGCAACUTT 32.64 5091-5109 AD-14899 829 AAGGGCGUGGAGGCUUUUUTT 830 AAAAAGCCUCCACGCCCUUTT 61.71 5093-5111 AD-14900 831 GUAAGGGCGUGGAGGCUUUTT 832 AAAGCCUCCACGCCCUUACTT 31.01 4259-4277 AD-14901 833 GACCCUAAAGACUUUCCUGTT 834 CAGGAAAGUCUUUAGGGUCTT 31.47 3901-3919 AD-14902 835 AGGAGCAUGACUUUAACCCTT 836 GGGUUAAAGUCAUGCUCCUTT 76.99 4757-4775 AD-14903 837 UGUGAUUUUCCUCCUAAUUTT 838 AAUUAGGAGGAAAAUCACATT 20.55 4758-4776 AD-14904 839 UUGUGAUUUUCCUCCUAAUTT 840 AUUAGGAGGAAAAUCACAATT 22.65 4562-4580 AD-14905 841 GACUUAACCCAAGAAGCUCTT 842 GAGCUUCUUGGGUUAAGUCTT 56.98 4585-4603 AD-14906 843 GCUUCAGACAAUGGUUUGGTT 844 CCAAACCAUUGUCUGAAGCTT 34.20 4587-4605 AD-14907 845 UUGCUUCAGACAAUGGUUUTT 846 AAACCAUUGUCUGAAGCAATT 28.59 4588-4606 AD-14908 847 AUUGCUUCAGACAAUGGUUTT 848 AACCAUUGUCUGAAGCAAUTT 34.08 4591-4609 AD-14909 849 UUGAUUGCUUCAGACAAUGTT 850 CAUUGUCUGAAGCAAUCAATT 76.57 5003-5021 AD-14910 851 GACAAAGUGCUGAAUAGGGTT 852 CCCUAUUCAGCACUUUGCCTT 46.50 4165-4183 AD-14911 853 AAGAAAAAGCUCAAAUUUUTT 854 AAAAUUUGAGCUUUUUCUUTT 29.62 4166-4184 AD-14912 855 AAAGAAAAAGCUCAAAUUUTT 856 AAAUUUGAGCUUUUUCUUUTT 22.27 4263-4281 AD-14913 857 AGAAGACCCUAAAGACUUUTT 858 AAAGUCUUUAGGGUCUUCUTT 59.80 4274-4292 AD-14914 859 AAAAAAAAGGUAGAAGACCTT 860 GGUCUUCUACCUUUUUUUUTT 93.21 4266-4284 AD-14915 861 GGUAGAAGACCCUAAAGACTT 862 GUCUUUAGGGUCUUCUACCTT 25.99 4272-4290 AD-14916 863 AAAAAAGGUAGAAGACCCUTT 864 AGGGUCUUCUACCUUUUUUTT 48.20 4271-4289 AD-14917 865 AAAAAGGUAGAAGACCCUATT 866 UAGGGUCUUCUACCUUUUUTT 41.03 4559-4577 AD-14918 867 UUAACCCAAGAAGCUCUUCTT 868 GAAGAGCUUCUUGGGUUAATT 110.62 4789-4807 AD-14919 869 AUUUUGGUACAUGGAAUAGTT 870 CUAUUCCAUGUACCAAAAUTT 73.66 4998-5016 AD-14920 871 AGUGCUGAAUAGGGAGGAATT 872 UUCCUCCCUAUUCAGCACUTT 19.80 5070-5088 AD-14921 873 GAGGCCAGGGAAAUUCCCUTT 874 AGGGAAUUUCCCUGGCCUCTT 33.13 4158-4176 AD-14922 875 AGCUCAAAUUUUAUAUAAGTT 876 CUUAUAUAAAAUUUGAGCUTT 52.94 5065-5083 AD-14923 877 CAGGGAAAUUCCCUUGUUUTT 878 AAACAAGGGAAUUUCCCUGTT 33.77 2872-2890 AD-14924 879 UUGUGCAGUGGAAAGAAAGTT 880 CUUUCUUUCCACUGCACAATT 64.47 4782-4800 AD-14925 881 UACAUGGAAUAGUUCAGAGTT 882 CUCUGAACUAUUCCAUGUATT 97.16 4783-4801 AD-14926 883 GUACAUGGAAUAGUUCAGATT 884 UCUGAACUAUUCCAUGUACTT 27.29 5064-5082 AD-14927 885 AGGGAAAUUCCCUUGUUUUTT 886 AAAACAAGGGAAUUUCCCUTT 27.02 5071-5089 AD-14928 887 GGAGGCCAGGGAAAUUCCCTT 888 GGGAAUUUCCCUGGCCUCCTT 76.75 3951-3969 AD-14929 889 GUGUAGACAGCCAUAUGCATT 890 UGCAUAUGGCUGUCUACACTT 32.92 3949-3967 AD-14930 891 GUAGACAGCCAUAUGCAGUTT 892 ACUGCAUAUGGCUGUCUACTT 31.00 4355-4373 AD-14931 893 GAAGACCUGUUUUGCCAUGTT 894 CAUGGCAAAACAGGUCUUCTT 31.36 4363-4381 AD-14932 895 AGUGGGAUGAAGACCUGUUTT 896 AACAGGUCUUCAUCCCACUTT 32.42 4356-4374 AD-14933 897 UGAAGACCUGUUUUGCCAUTT 898 AUGGCAAAACAGGUCUUCATT 39.94 4361-4379 AD-14934 899 UGGGAUGAAGACCUGUUUUTT 900 AAAACAGGUCUUCAUCCCATT 42.94 4560-4578 AD-14935 901 CUUAACCCAAGAAGCUCUUTT 902 AAGAGCUUCUUGGGUUAAGTT 47.74 2873-2891 AD-14936 903 AUUGUGCAGUGGAAAGAAATT 904 UUUCUUUCCACUGCACAAUTT 35.21 4730-4748 AD-14937 905 CUUUAUUGCAAGGAAUGGCTT 906 GCCAUUCCUUGCAAUAAAGTT 89.25 3899-3917 AD-14938 907 GAGCAUGACUUUAACCCAGTT 908 CUGGGUUAAAGUCAUGCUCTT 29.38 4756-4774 AD-14939 909 GUGAUUUUCCUCCUAAUUCTT 910 GAAUUAGGAGGAAAAUCACTT 26.45 4590-4608 AD-14940 911 UGAUUGCUUCAGACAAUGGTT 912 CCAUUGUCUGAAGCAAUCATT 77.50 4159-4177 AD-14941 913 AAGCUCAAAUUUUAUAUAATT 914 UUAUAUAAAAUUUGAGCUUTT 36.40 2743-2761 AD-14942 915 CUGGACAUGGAUCAAGCACTT 916 GUGCUUGAUCCAUGUCCAGTT 65.11 4155-4173 AD-14943 917 UCAAAUUUUAUAUAAGAAATT 918 UUUCUUAUAUAAAAUUUGATT 89.96 2871-2889 AD-14944 919 UGUGCAGUGGAAAGAAAGGTT 920 CCUUUCUUUCCACUGCACATT 48.98 4786-4804 AD-14945 921 UUGGUACAUGGAAUAGUUCTT 922 GAACUAUUCCAUGUACCAATT 43.45 4364-4382 AD-14946 923 AAGUGGGAUGAAGACCUGUTT 924 ACAGGUCUUCAUCCCACUUTT 41.25 4359-4377 AD-14947 925 GGAUGAAGACCUGUUUUGCTT 926 GCAAAACAGGUCUUCAUCCTT 42.10 2744-2762 AD-14948 927 UCUGGACAUGGAUCAAGCATT 928 UGCUUGAUCCAUGUCCAGATT 42.39 4787-4805 AD-14949 929 UUUGGUACAUGGAAUAGUUTT 930 AACUAUUCCAUGUACCAAATT 27.68

Relative siRNA activity SD of Residual (normalized SD of Relative residual luciferase to positive relative siRNA luciferase activity +/− control luc- siRNA activity +/− activity SD siRNA) activity SD 4.38 41 ± 4% 82.24 8.83 82 ± 9% 4.10 21 ± 4% 109.97 21.56 110 ± 22% 4.47 62 ± 4% 52.56 3.78 53 ± 4% 2.60 44 ± 3% 77.91 4.60 78 ± 5% 1.96 25 ± 2% 104.96 8.38 105 ± 8%  4.51 33 ± 5% 93.62 12.94  94 ± 13% 3.23 94 ± 3% 8.36 0.29  8 ± 0% 2.81 55 ± 3% 62.35 3.18 62 ± 3% 3.11 31 ± 3% 96.14 9.70  96 ± 10% 4.03 54 ± 4% 63.35 4.69 63 ± 5% 7.58 54 ± 8% 64.13 9.02 64 ± 9% 7.45 35 ± 7% 90.05 19.03  90 ± 19% 2.80 70 ± 3% 41.17 1.64 41 ± 2% 1.60 42 ± 2% 80.93 3.10 81 ± 3% 3.05 57 ± 3% 60.22 3.24 60 ± 3% 2.16 39 ± 2% 84.60 4.67 85 ± 5% 2.95 40 ± 3% 83.72 6.22 84 ± 6% 1.01 31 ± 1% 96.48 3.20 96 ± 3% 2.74 28 ± 3% 99.93 9.72 100 ± 10% 2.83 67 ± 3% 45.30 1.90 45 ± 2% 1.30 36 ± 1% 88.85 3.21 89 ± 3% 6.77 49 ± 7% 78.14 10.71  78 ± 11% 5.32 74 ± 5% 40.09 2.88 40 ± 3% 10.47  51 ± 10% 75.91 15.63  76 ± 16% 3.55 73 ± 4% 42.32 2.07 42 ± 2% 7.41 35 ± 7% 100.63 21.40 101 ± 21% 6.31 49 ± 6% 79.24 10.27  79 ± 10% 5.53 39 ± 6% 94.08 13.31  94 ± 13% 5.89 46 ± 6% 84.01 10.85  84 ± 11% 4.10 46 ± 4% 84.04 7.56 84 ± 8% 3.64 33 ± 4% 103.26 11.36 103 ± 11% 5.72 37 ± 6% 96.69 14.78  97 ± 15% 4.41 42 ± 4% 88.96 9.26 89 ± 9% 3.00 47 ± 3% 82.47 5.31 82 ± 5% 8.67 71 ± 9% 44.35 5.40 44 ± 5% 6.21 65 ± 6% 54.74 5.26 55 ± 5% 8.91 60 ± 9% 61.07 9.00 61 ± 9% 0.82 32 ± 1% 104.27 2.63 104 ± 3%  2.86 23 ± 3% 118.94 14.81 119 ± 15% 9.43 57 ± 9% 66.41 10.99  66 ± 11% 8.74 30 ± 9% 108.24 31.65 108 ± 32% 6.57 43 ± 7% 88.58 13.66  89 ± 14% 1.35 67 ± 1% 50.86 1.03 51 ± 1% 3.32 49 ± 3% 78.89 5.35 79 ± 5% 2.06 28 ± 2% 111.57 8.29 112 ± 8%  6.24 39 ± 6% 94.53 15.22  95 ± 15% 8.60 33 ± 9% 103.70 27.17 104 ± 27% 1.70 47 ± 2% 81.89 2.96 82 ± 3% 4.90 44 ± 5% 87.06 9.79  87 ± 10% 4.34 36 ± 4% 99.51 12.15 100 ± 12% 3.51 38 ± 4% 95.38 8.75 95 ± 9% 5.92 91 ± 6% 14.13 0.92 14 ± 1% 3.27 83 ± 3% 25.68 1.01 26 ± 1% 3.61 55 ± 4% 69.38 4.55 69 ± 5% 5.78 31 ± 6% 106.56 19.89 107 ± 20% 3.15 29 ± 3% 109.70 11.95 110 ± 12% 3.70 67 ± 4% 50.35 2.76 50 ± 3% 4.72 67 ± 5% 51.21 3.61 51 ± 4% 5.72 33 ± 6% 103.04 17.71 103 ± 18% 4.57 39 ± 5% 93.96 10.97  94 ± 11% 5.35 91 ± 5% 13.58 0.80 14 ± 1% 7.94 34 ± 8% 101.67 23.64 102 ± 24% 6.54 30 ± 7% 107.96 23.48 108 ± 23% 4.27 32 ± 4% 104.31 13.73 104 ± 14% 3.11 36 ± 3% 99.41 8.67 99 ± 9% 7.28 28 ± 7% 110.76 28.52 111 ± 29% 3.96 30 ± 4% 107.63 14.08 108 ± 14% 4.46 32 ± 4% 105.63 14.91 106 ± 15% 5.71 30 ± 6% 107.91 20.46 108 ± 20% 6.82 55 ± 7% 69.06 8.52 69 ± 9% 5.99 45 ± 6% 84.51 11.19  85 ± 11% 7.01 78 ± 7% 34.01 3.06 34 ± 3% 3.56 30 ± 4% 108.78 13.09 109 ± 13% 3.18 65 ± 3% 53.97 2.64 54 ± 3% 4.63 64 ± 5% 55.53 4.02 56 ± 4% 2.89 38 ± 3% 95.99 7.33 96 ± 7% 5.60 29 ± 6% 109.82 21.30 110 ± 21% 4.87 93 ± 5% 10.97 0.58 11 ± 1% 3.69 75 ± 4% 37.97 1.86 38 ± 2% 6.22 73 ± 6% 41.57 3.54 42 ± 4% 9.34 86 ± 9% 21.02 2.27 21 ± 2% 10.46  97 ± 10% 5.40 0.59  5 ± 1% 3.41 33 ± 3% 104.03 10.89 104 ± 11% 7.66 103 ± 8%  −4.18 0.31 −4 ± 0% 5.66 92 ± 6% 11.66 0.71 12 ± 1% 16.38  63 ± 16% 46.00 11.88  46 ± 12% 15.21  46 ± 15% 67.99 22.49  68 ± 22% 16.03  41 ± 16% 74.86 29.60  75 ± 30% 3.66 117 ± 4%  −21.52 0.67 −22 ± 1%  21.12  55 ± 21% 56.93 21.95  57 ± 22% 10.81  67 ± 11% 41.46 6.68 41 ± 7% 11.90  72 ± 12% 35.85 5.97 36 ± 6% 16.37  58 ± 16% 52.81 14.89  53 ± 15% 5.43 93 ± 5% 8.36 0.49  8 ± 0% 4.85 109 ± 5%  −11.13 0.50 −11 ± 0%  10.06 107 ± 10% −8.41 0.79 −8 ± 1% 6.68 37 ± 7% 79.23 14.28  79 ± 14% 7.54 36 ± 8% 80.53 16.84  81 ± 17% 5.90 39 ± 6% 77.40 11.86  77 ± 12% 8.91 111 ± 9%  −13.67 1.10 −14 ± 1%  5.51 35 ± 6% 82.04 12.98  82 ± 13% 6.04 24 ± 6% 95.99 24.41  96 ± 24% 5.29 27 ± 5% 91.30 17.57  91 ± 18% 4.70 93 ± 5% 8.67 0.44  9 ± 0% 8.26 82 ± 8% 23.01 2.33 23 ± 2% 5.29 78 ± 5% 27.83 1.89 28 ± 2% 4.95 44 ± 5% 69.99 7.81 70 ± 8% 5.08 46 ± 5% 67.46 7.38 67 ± 7% 6.70 36 ± 7% 81.17 15.31  81 ± 15% 1.13 36 ± 1% 102.63 3.22 103 ± 3%  6.75 68 ± 7% 51.41 5.11 51 ± 5% 3.07 69 ± 3% 49.05 2.17 49 ± 2% 6.88 29 ± 7% 113.79 26.89 114 ± 27% 7.07 36 ± 7% 102.68 20.14 103 ± 20% 7.93 34 ± 8% 106.57 25.15 107 ± 25% 8.76 51 ± 9% 79.04 13.64  79 ± 14% 7.26 54 ± 7% 74.49 10.09  74 ± 10% 9.34 39 ± 9% 97.82 23.38  98 ± 23% 6.85 63 ± 7% 59.75 6.52 60 ± 7% 1.86 87 ± 2% 20.12 0.43 20 ± 0% 4.02 80 ± 4% 32.19 1.62 32 ± 2% 4.49 30 ± 4% 111.64 16.46 112 ± 16% 5.07 33 ± 5% 107.26 16.38 107 ± 16% 3.98 26 ± 4% 118.39 17.96 118 ± 18% 1.24 37 ± 1% 101.57 3.44 102 ± 3%  3.13 33 ± 3% 107.30 10.12 107 ± 10% 4.56 30 ± 5% 112.52 17.16 113 ± 17% 3.66 28 ± 4% 115.20 14.91 115 ± 15% 3.04 50 ± 3% 79.67 4.81 80 ± 5% 5.11 39 ± 5% 97.32 12.63  97 ± 13% 4.12 35 ± 4% 104.82 12.43 105 ± 12% 1.79 32 ± 2% 108.93 6.07 109 ± 6%  4.87 102 ± 5%  −2.85 0.14 −3 ± 0% 4.39 81 ± 4% 30.80 1.67 31 ± 2% 1.88 31 ± 2% 111.18 6.81 111 ± 7%  2.84 57 ± 3% 68.42 3.39 68 ± 3% 3.35 70 ± 3% 47.79 2.28 48 ± 2% 7.72 79 ± 8% 33.66 3.29 34 ± 3% 2.46 22 ± 2% 125.78 14.28 126 ± 14% 1.71 28 ± 2% 116.13 7.17 116 ± 7%  2.94 34 ± 3% 105.93 9.16 106 ± 9%  3.22 41 ± 3% 95.33 7.56 95 ± 8% 5.94 36 ± 6% 103.18 17.14 103 ± 17% 7.65 47 ± 8% 84.45 13.63  84 ± 14% 3.94 37 ± 4% 100.76 10.67 101 ± 11% 7.26 43 ± 7% 91.61 15.50  92 ± 15% 4.06 33 ± 4% 108.24 13.50 108 ± 14% 2.98 83 ± 3% 27.15 0.97 27 ± 1% 2.94 59 ± 3% 65.04 3.22 65 ± 3% 5.52 22 ± 6% 125.32 31.52 125 ± 32% 2.19 73 ± 2% 43.84 1.32 44 ± 1% 7.07 24 ± 7% 121.32 35.11 121 ± 35% 5.08 34 ± 5% 106.20 15.95 106 ± 16% 4.46 22 ± 4% 125.73 25.84 126 ± 26% 5.01 27 ± 5% 117.20 21.73 117 ± 22% 2.72 29 ± 3% 113.92 10.67 114 ± 11% 4.87 33 ± 5% 108.14 16.13 108 ± 16% 4.59 62 ± 5% 61.47 4.57 61 ± 5% 2.84 31 ± 3% 110.75 10.14 111 ± 10% 1.57 31 ± 2% 110.01 5.49 110 ± 5%  0.55 77 ± 1% 36.95 0.26 37 ± 0% 3.55 21 ± 4% 127.55 22.05 128 ± 22% 6.87 23 ± 7% 124.18 37.68 124 ± 38% 4.94 57 ± 5% 69.07 5.99 69 ± 6% 3.66 34 ± 4% 105.63 11.29 106 ± 11% 8.12 29 ± 8% 114.64 32.56 115 ± 33% 3.36 34 ± 3% 105.82 10.44 106 ± 10% 2.33 77 ± 2% 37.61 1.15 38 ± 1% 4.14 46 ± 4% 85.89 7.64 86 ± 8% 2.02 30 ± 2% 112.99 7.69 113 ± 8%  0.48 22 ± 0% 124.78 2.69 125 ± 3%  2.85 60 ± 3% 64.53 3.08 65 ± 3% 5.10 93 ± 5% 10.90 0.60 11 ± 1% 4.45 26 ± 4% 118.82 20.34 119 ± 20% 1.46 48 ± 1% 83.16 2.51 83 ± 3% 3.07 41 ± 3% 94.67 7.08 95 ± 7% 6.34 111 ± 6%  −17.04 0.98 −17 ± 1%  3.68 74 ± 4% 42.29 2.11 42 ± 2% 1.72 20 ± 2% 128.75 11.20 129 ± 11% 1.14 33 ± 1% 107.34 3.71 107 ± 4%  6.99 53 ± 7% 63.41 8.37 63 ± 8% 8.92 34 ± 9% 89.23 23.56  89 ± 24% 10.96  64 ± 11% 47.86 8.13 48 ± 8% 7.57 97 ± 8% 3.83 0.30  4 ± 0% 8.79 27 ± 9% 97.96 31.56  98 ± 32% 10.01  27 ± 10% 98.33 36.42  98 ± 36% 4.78 77 ± 5% 31.32 1.95 31 ± 2% 9.44 33 ± 9% 90.38 25.93  90 ± 26% 9.40 31 ± 9% 92.97 28.21  93 ± 28% 8.73 31 ± 9% 92.48 25.74  92 ± 26% 9.01 32 ± 9% 91.05 25.29  91 ± 25% 6.96 40 ± 7% 80.92 14.10  81 ± 14% 7.66 43 ± 8% 76.88 13.71  77 ± 14% 8.48 48 ± 8% 70.41 12.51  70 ± 13% 4.02 35 ± 4% 87.29 9.97  87 ± 10% 3.53 89 ± 4% 14.48 0.57 14 ± 1% 6.46 29 ± 6% 95.15 20.91  95 ± 21% 8.33 26 ± 8% 99.09 31.21  99 ± 31% 6.51 78 ± 7% 30.31 2.55 30 ± 3% 10.76  36 ± 11% 85.68 25.32  86 ± 25% 5.84 65 ± 6% 47.01 4.22 47 ± 4% 4.69 90 ± 5% 13.52 0.71 14 ± 1% 5.85 49 ± 6% 68.74 8.21 69 ± 8% 3.29 43 ± 3% 76.18 5.77 76 ± 6% 1.26 41 ± 1% 79.15 2.43 79 ± 2% 7.34 42 ± 7% 78.01 13.60  78 ± 14% 5.75 42 ± 6% 77.62 10.54  78 ± 11% 5.79 28 ± 6% 97.44 20.39  97 ± 20%

siRNAs targeting JCV transcripts for primary screen Description of chemistries: a b c d sense strand antisense strand position SEQ SEQ duplex in ID ID name chemistry consensus NO: sequence (5′-3′) NO: sequence (5′-3′) AD-12598 a 1426-1444 1 AcuuuuAGGGuuGuAcGGGTsT 2 CcCGuAcAACCCuAAAAGUTsT AD-12708 b 1426-1444 3 AcuuuuAGGGuuGuAcGGGTsT 4 CCCGuAcAACCCuAAAAGUTsT AD-12599 a 1427-1445 5 cuuuuAGGGuuGuAcGGGATsT 6 UcCCGuAcAACCCuAAAAGTsT AD-12709 b 1427-1445 7 cuuuuAGGGuuGuAcGGGATsT 8 UCCCGuAcAACCCuAAAAGTsT AD-12600 a 2026-2044 9 cAGAGcAcAAGGcGuAccuTsT 10 AgGuACGCCUUGUGCUCUGTsT AD-12710 b 2026-2044 11 cAGAGcAcAAGGcGuAccuTsT 12 AGGuACGCCUUGUGCUCUGTsT AD-12784 c 2026-2044 13 caGAGcAcAAGGcGuAccuTsT 14 AGGuACGCCUUGUGCUCUGTsT AD-12832 d 2026-2044 15 caGAGcAcAAGGcGuAccuTsT 16 AgGuACGCCUUGUGCUCUGTsT AD-12601 a 1431-1449 17 uAGGGuuGuAcGGGAcuGuTsT 18 AcAGUCCCGuAcAACCCuATsT AD-12785 c 1431-1449 19 uaGGGuuGuAcGGGAcuGuTsT 20 AcAGUCCCGuAcAACCCuATsT AD-12602 a 1432-1450 21 AGGGuuGuAcGGGAcuGuATsT 22 uacAGUCCCGuAcAACCCUTsT AD-12711 b 1432-1450 23 AGGGuuGuAcGGGAcuGuATsT 24 uAcAGUCCCGuAcAACCCUTsT AD-12786 c 1432-1450 25 AgGGuuGuACGGGACuGuATsT 26 uACAGUCCCGuACAACCCUTsT AD-12833 d 1432-1450 27 AgGGuuGuACGGGACuGuATsT 28 uaCAGUCCCGuACAACCCUTsT AD-12603 a 1436-1454 29 uuGuACGGGACuGuAACACTsT 30 GuGUuACAGUCCCGuACAATsT AD-12712 b 1436-1454 31 uuGuACGGGACuGuAACACTsT 32 GUGUuACAGUCCCGuACAATsT AD-12604 a 4794-4812 33 GaauGAuuuuGGuAaAuGGTsT 34 CCAUGuACCAAAAUCAGGCTsT AD-12605 a 5099-5117 35 GAAGuAGuAAGGGCGuGGATsT 36 UCCACGCCCUuACuACUUCTsT AD-12713 b 5099-5117 37 GAAGuAGuAAGGGCGuGGATsT 38 UCCACGCCCUuACuACUUCTsT AD-12787 c 5099-5117 39 GaAGuAGuAAGGGCGuGGATsT 40 UCCACGCCCUuACuACUUCTST AD-12834 d 5099-5117 41 GaAGuAGuAAGGGCGuGGATsT 42 UCCACGCCCUuACuACUUCTsT AD-12606 a 713-731 43 AuAGGaauuAauaauGAAATsT 44 UuUCAGGAGuAAGGCCuAUTsT AD-12714 b 713-131 45 AuAGGccuuAcuccuGAAATsT 46 UUUcAGGAGuAAGGCCuAUTsT AD-12607 a 3946-3964 47 GAcAGccAuAuGcAGuAGuTsT 48 AcuACUGcAuAUGGCUGUCTsT AD-12715 b 3946-3964 49 GAcAGccAuAuGcAGuGuTsT 50 ACuACUGcAuAUGGCUGUCTsT AD-12788 c 3946-3964 51 GacAGccAuAuGcAGuAGuTsT 52 ACuACUGcAuAUGGCUGUCTsT AD-12835 d 3946-3964 53 GacAGccAuAuGcAGUGUTsT 54 AcuACUGcAuAUGGCUGUCTsT AD-12608 a 1128-1146 55 AAAcuAcuuGGGcAAuAGuTsT 56 AcuAUUGCCcAAGuAGUUUTsT AD-12716 b 1128-1146 57 AAAcuAcuuGGGcAAuAGuTsT 58 ACuAUUGCCcAAGuAGUUUTsT AD-12789 c 1128-1146 59 AaAcuAcuuGGGcAAuAGuTsT 60 ACuAUUGCCcAAGuAGUUUTsT AD-12836 d 1128-1146 61 AaAcuAcuGGGcAAoAGuTsT 62 AcuAUUGCCcAAGuAGUUUTsT AD-12609 a 525-543 63 ucAGGuucAuGGGuGccGcTsT 64 GcGGcACCcAUGAACCUGATsT AD-12717 b 525-543 65 ucAGGuucAuGGGuGccGcTsT 66 GcGGcACCcAUGAACCUGATsT AD-12610 a 5096-5114 67 GuAGuAAGGGcGuGGaGGcTsT 68 GcCUCcAcGCCCUuACuATsT AD-12718 b 5096-5114 69 GuAGuAAGGGcGuGGaGGcTsT 70 GcCUCcAcGCCCUuACuATsT AD-12611 a 4727-4745 71 uAuuGcAAGGAAuGGccuATsT 72 uaGGCcAUUCCUUUGcAAuATsT AD-12719 b 4727-4745 73 uAuuGcAAGGAAuGGccuATsT 74 uAGGCcAUUCCUUUGcAAuATsT AD-12790 c 4727-4745 75 uAuuGcAAGGAAuGGccuATsT 76 uAGGCcAUUCCUUUGcAAuATsT AD-12837 d 4727-4745 77 uAuuGcAAGGAAuGGccuATsT 78 uaGGCcAUUCCUUUGcAAuATsT AD-12612 a 5097-5115 79 AGuAGuAAGGGcGuGGAGGTsT 80 CcUCcACGCCCUuACuACUTsT AD-12720 b 5097-5115 81 AGuAGuAAGGGcGuGGAGGTsT 82 CCUCcACGCCCUuACuACUTsT AD-12791 c 5097-5115 83 AguAGuAAGGGcGuGGAGGTsT 84 CCUCcACGCCCUuACuACUTsT AD-12838 d 5097-5115 85 AguAGuAAGGGcGuGGAGGTsT 86 CcUCcACGCCCUuACuACUTsT AD-12613 a 4601-4619 87 uGcuAuuGcuuuGAuuGcuTsT 88 AgcAAUcAAAGcAAuAGcATsT AD-12721 b 4601-4619 89 uGcuAuuGcuuuGAuuGcuTsT 90 AGcAAUcAAAGcAAuAGcATsT AD-12792 c 4601-4619 91 ugcuAuuGcuuuGAuuGcuTsT 92 AGcAAUcAAAGcAAuAGcATsT AD-12839 d 4601-4619 93 ugcuAuuGcuuuGAuuGcuTsT 94 AgcAAUcAAAGcAAuAGcATsT AD-12614 a 4600-4618 95 GcuAuuGcuuuGAuuGcuuTsT 96 AaGcAAUcAAAGcAAuAGCTsT AD-12722 b 4600-4618 97 GcuAuuGcuuuGAuuGcuuTsT 98 AaGcAAUcAAAGcAAuAGCTsT AD-12615 a 1421-1439 99 ccuuuAcuuuuAGGGuuGuTsT 100 AcAACCCuAAAAGuAAAGGTsT AD-12616 a 1424-1442 101 uuAcuuuuAGGGuuGuAcGTsT 102 CguAcAACCCuAAAAGuAATsT AD-12723 b 1424-1442 103 uuAcuuuuAGGGuuGuAcGTsT 104 CguAcAACCCuAAAAGuAATsT AD-12617 a 1403-1421 105 GcuccucAAuGGAuGuuGcTsT 106 GcAAcAUCcAUUGAGGAGCTsT AD-12618 a 1534-1552 107 uuAuAAGAGGAGGAGuAGATsT 108 UcuACUCCUCCUCUuAUAATsT AD-12724 b 1534-1552 109 uuAuAAGAGGAGGAGuAGATsT 110 UcuACUCCUCCUCUuAUAATsT AD-12619 a 5098-5116 111 AAGuAGuAAGGGcGuGGAGTsT 112 CuCcAcGCCCUuACuACUUTsT AD-12725 b 5098-5116 113 AAGuAGuAAGGGcGuGGAGTsT 114 CuCcAcGCCCUuACuACUUTsT AD-12793 c 5098-5116 115 AAGuAGuAAGGGcGuGGAGTsT 116 CuCcAcGCCCUuACuACUUTsT AD-12840 d 5098-5116 117 AAGuAGuAAGGGcGuGGAGTsT 118 CuCcAcGCCCUuACuACUUTsT AD-12620 a 1430-1448 119 uuAGGGuuGuAcGGGAcuGTsT 120 caGUCCCGuAcAACCCuAATsT AD-12726 b 1430-1448 121 uuAGGGuuGuAcGGGAcuGTsT 122 caGUCCCGuAcAACCCuAATsT AD-12621 a 1701-1719 123 GAcAuGcuuccuuGuuAcATsT 124 UguAAcAAGGAAGcAUGUCTsT AD-12727 b 1701-1719 125 GAcAuGcuuccuuGuuAcATsT 126 UGuAAcAAGGAAGcAUGUCTsT AD-12794 c 1701-1719 127 GAcAuGcuuccuuGuuAcATsT 128 UGuAAcAAGGAAGcAUGUCTsT AD-12841 d 1701-1719 129 GAcAuGcuuccuuGuuAcATsT 130 UguAAcAAGGAAGcAUGUCTsT AD-12622 a 2066-2084 131 uGuuGAAuGuuGGGuuccuTsT 132 AgGAAcCcAAcAUUcAAcATsT AD-12728 b 2066-2084 133 uGuuGAAuGuuGGGuuccuTsT 134 AGGAAcCcAAcAUUcAAcATsT AD-12795 c 2066-2084 135 uguuGAAuGuuGGGuuccuTsT 136 AGGAAcCcAAcAUUcAAcATsT AD-12842 d 2066-2084 137 uguuGAAuGuuGGGuuccuTsT 138 AgGAAcCcAAcAUUcAAcATsT AD-12623 a 4561-4579 139 AcuuAAcccAAGAAGcucuTsT 140 AgAGCUUCUUGGGUuAAGUTsT AD-12729 b 4561-4579 141 AcuuAAcccAAGAAGcucuTsT 142 AgAGCUUCUUGGGUuAAGUTsT AD-12624 a 4797-4815 143 ucAGccuGAuuuuGGuAcATsT 144 UguACcAaAAUcAGGCUGATsT AD-12730 b 4797-4815 145 ucAGccuGAuuuuGGuAcATsT 146 UGuACcAaAAUcAGGCUGATsT AD-12625 a 1428-1446 147 uuuuAGGGuuGuAcGGGAcTsT 148 GuCCCGuAcAACCCuAAAATsT AD-12731 b 1428-1446 149 uuuuAGGGuuGuAcGGGAcTsT 150 GUCCCGuAcAACCCuAAAATsT AD-12626 a 1429-1447 151 uuuAGGGuuGuAcGGGAcuTsT 152 AgUCCCGuAcAACCCuAAATsT AD-12732 b 1429-1447 153 uuuAGGGuuGuAcGGGAcuTsT 154 AAGUCCCGuAcAACCCuAAATsT AD-12627 a 662-680 155 uccccuuGcuAcuGuAGAGGTsT 156 CcUCuAcAGuAGcAAGGGATsT AD-12733 b 662-6 157 uccccuuGcuAcuGuAGAGGTsT 158 CCUCuAcAGuAGcAAGGGATsT AD-12628 a 663-681 159 cccuuGcuAcuGuAGAGGGTsT 160 CcUCuAcAGuAGcAAGGGATsT AD-12734 b 663-681 161 cccuuGcuAcuGuAGAGGGTsT 162 CCUCuAcAGuAGcAAGGGATsT AD-12629 a 1402-1420 163 uGcuccucAAuGGAuGuuGTsT 164 caAcAUCcAUUGAGGAGcATsT AD-12735 b 1402-1420 165 uGcuccucAAuGGAuGuuGTsT 166 caAcAUCcAUUGAGGAGcATsT AD-12796 c 1402-1420 167 ugcuccucAAuGGAuGuuGTsT 168 caAcAUCcAUUGAGGAGcATsT AD-12843 d 1402-1420 169 ugcuccucAAuGGAuGuuGTsT 170 caAcAUCcAUUGAGGAGcATsT AD-12630 a 1398-1416 171 GAucuGcuccucAAuGGAuTsT 172 AuCcAUUGAGGAGcAGAUCTsT AD-12736 b 1398-1416 173 GAucuGcuccucAAuGGAuTsT 174 AUCcAUUGAGGAGcAGAUCTsT AD-12797 c 1398-1416 175 GaucuGcuccucAAuGGAuTsT 176 AUCcAUUGAGGAGcAGAUCTsT AD-12844 d 1398-1416 177 GaucuGcuccucAAuGGAuTsT 178 AuCcAUUGAGGAGcAGAUCTsT AD-12631 a 1399-1417 179 AucuGcuccucAauGGAuGTsT 180 caUCcAUUGAGGAGcAGAUTsT AD-12737 b 1399-1417 181 AucuGcuccucAauGGAuGTsT 182 cAUCcAUUGAGGAGcAGAUTsT AD-12632 a 1400-1418 183 ucuGcuccucAAuGGAuGuTsT 184 AcAUCcAUUGAGGAGcAGATsT AD-12633 a 1401-1419 185 cuGcuccucAAuGGAuGuuTsT 186 AacAUCcAUUGAGGAGcAGTsT AD-12738 b 1401-1419 187 cuGcuccucAAuGGAuGuuTsT 188 AacAUCcAUUGAGGAGcAGTsT AD-12634 a 1435-1453 189 GuuGuAcGGGAcuGuAAcATsT 190 UgUuAcAGUCCCGuAcAACTsT AD-12739 b 1435-1453 191 GuuGuAcGGGAcuGuAAcATsT 192 UgUuAcAGUCCCGuAcAACTsT AD-12635 a 1437-1455 193 uGuAcGGGAcuGuAAcAccTsT 194 GgUGUuAcAGUCCCGuAcATsT AD-12740 b 1437-1455 195 uGuAcGGGAcuGuAAcAccTsT 196 GGUGUuAcAGUCCCGuAcATsT AD-12798 c 1437-1455 197 uguAcGGGAcuGuAAcAccTsT 198 GGUGUuAcAGUCCCGuAcATsT AD-12845 d 1437-1455 199 uguAcGGGAcuGuAAcAccTsT 200 GgUGUuAcAGUCCCGuAcATsT AD-12636 a 1438-1456 201 GuAcGGGAcuGuAAcAccuTsT 202 AgGUGUuAcAGUCCCGuACTsT AD-12741 b 1438-1456 203 GuAcGGGAcuGuAAcAccuTsT 204 AgGUGUuAcAGUCCCGuACTsT AD-12637 a 4796-4814 205 cAGccuGAuuuuGGuAcAuTsT 206 AuGuACcAAAAUcAGGCUGTsT AD-12742 b 4796-4814 207 cAGccuGAuuuuGGuAcAuTsT 208 AUGuACcAAAAUcAGGCUGTsT AD-12799 c 4796-4814 209 caGccuGAuuuuGGuAcAuTsT 210 AUGuACcAAAAUcAGGCUGTsT AD-12846 d 4796-4814 211 caGccuGAuuuuGGuAcAuTsT 212 AuGuACcAAAAUcAGGCUGTsT AD-12638 a 4992-5010 213 GAAuAGGGAGGAAuccAuGTsT 214 caUGGAUUCCUCCCuAUUCTsT AD-12743 b 4992-5010 215 GAAuAGGGAGGAAuccAuGTsT 216 cAUGGAUUCCUCCCuAUUCTsT AD-12800 c 4992-5010 217 GaAuAGGGAGGAAuccAuGTsT 218 cAUGGAUUCCUCCCuAUUCTsT AD-12847 d 4992-5010 219 GaAuAGGGAGGAAuccAuGTsT 220 caUGGAUUCCUCCCuAUUCTsT AD-12639 a 4999-5017 221 AAGuGcuGAAuAGGGAGGATsT 222 UcCUCCCuAUUcAGcACUUUTsT AD-12744 b 4999-5017 223 AAGuGcuGAAuAGGGAGGATsT 224 UcCUCCCuAUUcAGcACUUUTsT AD-12801 c 4999-5017 225 AaGuGcuGAAuAGGGAGGATsT 226 UcCUCCCuAUUcAGcACUUUTsT AD-12848 d 4999-5017 227 AaGuGcuGAAuAGGGAGGATsT 228 UcCUCCCuAUUcAGcACUUUTsT AD-12640 a 630-648 229 AGGcuGcuGcuAcuAuAGATsT 230 UcuAuAGuAGcAGcAGCCUTsT AD-12745 b 630-648 231 AGGcuGcuGcuAcuAuAGATsT 232 UCuAuAGuAGcAGcAGCCUTsT AD-12802 c 630-648 233 AgGcuGcuGcuAcuAuAGATsT 234 UCuAuAGuAGcAGcAGCCUTsT AD-12849 d 630-648 234 AgGcuGcuGcuAcuAuAGATsT 236 UcuAuAGuAGcAGcAGCCUTsT AD-12641 a 3947-3965 237 AGAcAGccAuAuGcAGuAGTsT 238 CuACUGcAuAUGGUGUCUTsT AD-12803 c 3947-3965 239 AGAcAGccAuAuGcAGuAGTsT 240 CuACUGcAuAUGGUGUCUTsT AD-12642 a 524-542 241 uucAGGuucAuGGGuGccGTsT 242 CgGcACCcAUGAACCUGAATsT AD-12746 b 524-542 243 uucAGGuucAuGGGuGccGTsT 244 CGGcACCcAUGAACCUGAATsT AD-12643 a 3948-3966 245 uAGAcAGccAuAuGcAGuATsT 246 uaCUGcAuAUGGCUGUCuATST AD-12747 b 3948-3966 247 uAGAcAGccAuAuGcAGuATsT 248 uACUGcAuAUGGCUGUCuATST AD-12804 c 3948-3966 249 uaGAcAGccAuAuGcAGuATsT 250 uACUGcAuAUGGCUGUCuATST AD-12850 d 3948-3966 251 uaGAcAGccAuAuGcAGuATsT 252 uaCUGcAuAUGGCUGUCuATST AD-12644 a 3900-3918 253 GGAGcAuGAcuuuAAcccATsT 254 UgGGUuAAAGUcAUGCUCCTsT AD-12748 b 3099-3918 255 GGAGcAuGAcuuuAAcccATsT 256 UGGGUuAAAGUcAUGCUCCTsT AD-12805 c 3099-3918 257 GgAGcAuGAcuuuAAcccATsT 258 UGGGUuAAAGUcAUGCUCCTsT AD-12851 d 3099-3918 259 GgAGcAuGAcuuuAAcccATsT 260 UgGGUuAAAGUcAUGCUCCTsT AD-12645 a 1417-1435 261 GuuGccuuuAcuuuuAGGGTsT 262 CcCuAAAGuAAAGGcAACCTsT AD-12749 b 1417-1435 263 GuuGccuuuAcuuuuAGGGTsT 264 CCCuAAAGuAAAGGcAACCTsT AD-12646 a 4565-4583 265 uGuGAcuuAAcccAAGAAGTsT 266 CuUCUUGGGUuAAGUcAcATsT AD-12750 b 4565-4583 267 uGuGAcuuAAcccAAGAAGTsT 268 CUUCUUGGGUuAAGUcAcATsT AD-12806 c 4565-4583 269 uguGAcuuAAcccAAGAAGTsT 270 CUUCUUGGGUuAAGUcAcATsT AD-12852 d 4565-4583 271 uguGAcuuAAcccAAGAAGTsT 272 CuUCUUGGGUuAAGUcAcATsT AD-12647 a 4598-4616 273 uAuuGcuuuGAuuGcuucATsT 274 UgAAGcAAUcAAAGcAAuATsT AD-12751 b 4598-4616 275 uAuuGcuuuGAuuGcuucATsT 276 UGAAGcAAUcAAAGcAAuATsT AD-12807 c 4598-4616 277 uauuGcuuuGAuuGcuucATsT 278 UGAAGcAAUcAAAGcAAuATsT AD-12853 d 4598-4616 279 uauuGcuuuGAuuGcuucATsT 280 UgAAGcAAUcAAAGcAAuATsT AD-12648 a 2060-2078 281 AuAuccuGuuGAAuGuuGGTsT 282 CcAacAUUcAAcAGGAuAUTsT AD-12649 a 4729-4747 283 uuuAuuGcAAGGAAuGGccTsT 284 GgCcAUUCCUUGcAAuAAATsT AD-12752 b 4729-4747 285 uuuAuuGcAAGGAAuGGccTsT 286 GGCcAUUCCUUGcAAuAAATsT AD-12650 a 1122-1140 287 uGGAAGAAAcuAcuuGGGcTsT 288 GcCcAAGuAGUUUCUUCcATsT AD-12753 b 1122-1140 289 uGGAAGAAAcuAcuuGGGcTsT 290 GCCcAAGuAGUUUCUUCcATsT AD-12808 c 1122-1140 291 ugGAAGAAAcuAcuuGGGcTsT 292 GCCcAAGuAGUUUCUUCcATsT AD-12854 d 1122-1140 293 ugGAAGAAAcuAcuuGGGcTsT 294 GcCcAAGuAGUUUCUUCcATsT AD-12651 a 4261-4279 295 AAGAcccuAAAGAcuuuccTsT 296 GgAAAGUCUUuAGGGUCUUTsT AD-12754 b 4261-4279 297 AAGAcccuAAAGAcuuuccTsT 298 GGAAAGUCUUuAGGGUCUUTsT AD-12809 c 4261-4279 299 AaGAcccuAAAGAcuuuccTsT 300 GGAAAGUCUUuAGGGUCUUTsT AD-12855 d 4261-4279 301 AaGAcccuAAAGAcuuuccTsT 302 GgAAAGUCUUuAGGGUCUUTsT AD-12652 a 1412-1430 303 uGGAuGuuGccuuuAcuuuTsT 304 AaAGuAAAGGcAAcAUCcATsT AD-12755 b 1412-1430 305 uGGAuGuuGccuuuAcuuuTsT 306 AAAGuAAAGGcAAcAUCcATsT AD-12810 c 1412-1430 307 ugGAuGuuGccuuuAcuuuTsT 308 AAAGuAAAGGcAAcAUCcATsT AD-12856 d 1412-1430 309 ugGAuGuuGccuuuAcuuuTsT 310 AaAGuAAAGGcAAcAUCcATsT AD-12653 a 4592-4610 311 uuuGAuuGcuucAGAcAAuTsT 312 AuUGUCUGAAGcAAUcAAATsT AD-12756 b 4592-4610 313 uuuGAuuGcuucAGAcAAuTsT 314 AuUGUCUGAAGcAAUcAAATsT AD-12654 a 4991-5009 315 AAuAGGGAGGAAuccAuGGTsT 316 CcAUGGAUUCCUCCCuAUUTsT AD-12811 c 4991-5009 317 AAuAGGGAGGAAuccAuGGTsT 318 CcAUGGAUUCCUCCCuAUUTsT AD-12655 a 5004-5022 319 GGAcAAAGuGcuGAAuAGGTsT 320 CcuAUUcAGcAcUUUGUCCTsT AD-12757 b 5004-5022 321 GGAcAAAGuGcuGAAuAGGTsT 322 CcuAUUcAGcAcUUUGUCCTsT AD-12812 c 5004-5022 323 GgAcAAAGuGcuGAAuAGGTsT 324 CCuAUUcAGcAcUUUGUCCTsT AD-12857 d 5004-5022 325 GgAcAAAGuGcuGAAuAGGTsT 326 CCuAUUcAGcAcUUUGUCCTsT AD-12656 a 5005-5023 327 uGGAcAAAGuGcuGAAuAGTsT 328 CuAUUcAGcACUUUGUCcATsT AD-12813 c 5005-5023 329 ugGAcAAAGuGcuGAAuAGTsT 330 CuAUUcAGcACUUUGUCcATsT AD-12657 a 654-672 331 AAAuuGcAucccuuGcuAcTsT 332 GuAGcAAGGGAUGcAAUUUTsT AD-12814 c 654-672 333 AaAuuGcAucccuuGcuAcTsT 334 GuAGcAAGGGAUGcAAUUUTsT AD-12658 a 659-677 335 GcAucccuuGcuAcuGuAGTsT 336 CuAcAGuAGcAAGGGAUGCTsT AD-12659 a 4273-4291 337 AAAAAAAGGuAGAAGAcccTsT 338 GgGUCUUCuACCUUUUUUUTsT AD-12758 b 4273-4291 339 AAAAAAAGGuAGAAGAcccTsT 340 GGGUCUUCuACCUUUUUUUTsT AD-12815 c 4273-4291 341 AaAAAAAGGuAGAAGAcccTsT 342 GGGUCUUCuACCUUUUUUUTsT AD-12858 d 4273-4291 343 AaAAAAAGGuAGAAGAcccTsT 344 GgGUCUUCuACCUUUUUUUTsT AD-12660 a 2025-2043 345 AcAGAGcAcAAGGcGuAccTsT 346 GguACGCCUUGUGCUCUGUTsT AD-12759 b 2025-2043 347 AcAGAGcAcAAGGcGuAccTsT 348 GGuACGCCUUGUGCUCUGUTsT AD-12661 a 4791-4809 349 uGAuuuuGGuAcAuGGAAuTsT 350 AuUCcAUGuACcAAAAUcATsT AD-12760 b 4791-4809 351 uGAuuuuGGuAcAuGGAAuTsT 352 AUUCcAUGuACcAAAAUcATsT AD-12816 c 4791-4809 353 ugAuuuuGGuAcAuGGAAuTsT 354 AUUCcAUGuACcAAAAUcATsT AD-12859 d 4791-4809 355 ugAuuuuGGuAcAuGGAAuTsT 356 AuUCcAUGuACcAAAAUcATsT AD-12662 a 1433-1451 357 GGGuuGuAcGGGAcuGuAATsT 358 UuAcAGUCCCGuAcAACCCTsT AD-12817 c 1433-1451 359 GgGuuGuAcGGGAcuGuAATsT 360 UuAcAGUCCCGuAcAACCCTsT AD-12663 a 1434-1452 361 GGuuGuAcGGGAcuGuAAcTsT 362 GuuAcAGUCCCGuAcAACCTsT AD-12761 b 1434-1452 363 GGuuGuAcGGGAcuGuAAcTsT 364 GUuAcAGUCCCGuAcAACCTsT AD-12818 c 1434-1452 365 GguuGuAcGGGAcuGuAAcTsT 366 GUuAcAGUCCCGuAcAACCTsT AD-12860 d 1434-1452 367 GguuGuAcGGGAcuGuAAcTsT 368 GuuAcAGUCCCGuAcAACCTsT AD-12664 a 1440-1458 369 AcGGGAcuGuAAcAccuGcTsT 370 GcAGGUGUuAcAGUCCCGUTsT AD-12665 a 1442-1460 371 GGGAcuGuAAcAccuGcucTsT 372 GaGcAGGUGUuAcAGUCCCTsT AD-12762 b 1442-1460 373 GGGAcuGuAAcAccuGcucTsT 374 GaGcAGGUGUuAcAGUCCCTsT AD-12819 c 1442-1460 375 GgGAcuGuAAcAccuGcucTsT 376 GAGcAGGUGUuAcAGUCCCTsT AD-12861 d 1442-1460 377 GgGAcuGuAAcAccuGcucTsT 378 GAGcAGGUGUuAcAGUCCCTsT AD-12666 a 1608-1626 379 AcuccAgAAAuGGGuGAccTsT 380 GgUcACCcAUUUCUGGAGUTsT AD-12763 b 1608-1626 381 AcuccAgAAAuGGGuGAccTsT 382 GGUcACCcAUUUCUGGAGUTsT AD-12667 a 4793-4811 383 ccuGAuuuuGGuAcAuGGATsT 384 UccAUGuACcAAAAUcAGGTsT AD-12764 b 4793-4811 385 ccuGAuuuuGGuAcAuGGATsT 386 UCcAUGuACcAAAAUcAGGTsT AD-12668 a 5001-5019 387 cAAAGuGcuGAAuAGGGAGTsT 388 CuCCCuAUUcAGcACUUUGTsT AD-12765 b 5001-5019 389 cAAAGuGcuGAAuAGGGAGTsT 390 CUCCCuAUUcAGcACUUUGTsT AD-12820 c 5001-5019 391 caAAGuGcuGAAuAGGGAGTsT 392 CUCCCuAUUcAGcACUUUGTsT AD-12862 d 5001-5019 393 caAAGuGcuGAAuAGGGAGTsT 394 CuCCCuAUUcAGcACUUUGTsT AD-12669 a 5066-5084 395 ccAGGGAAAuucccuuGuuTsT 396 AacAAGGGAAUUUCCCUGGTsT AD-12766 b 5066-5084 397 ccAGGGAAAuucccuuGuuTsT 398 AAcAAGGGAAUUUCCCUGGTsT AD-12670 a 5069-5087 399 AGGccAGGGAAAuucccuuTsT 400 AaGGGAAUUUCCCUGGCCUTsT AD-12767 b 5069-5087 401 AGGccAGGGAAAuucccuuTsT 402 AAGGGAAUUUCCCUGGCCUTsT AD-12821 c 5069-5087 403 AgGccAGGGAAAuucccuuTsT 404 AAGGGAAUUUCCCUGGCCUTsT AD-12863 d 5069-5087 405 AgGccAGGGAAAuucccuuTsT 406 AaGGGAAUUUCCCUGGCCUTsT AD-12671 a 564-582 407 uAGuuGcuAcuGuuucuGATsT 408 UcAGAAAcAGuAGcAACuATsT AD-12822 c 564-582 409 uAGuuGcuAcuGuuucuGATsT 410 UcAGAAAcAGuAGcAACuATsT AD-12672 a 633-651 411 cuGcuGcuAcuAuAGAAGuTsT 412 AcUUCuAuAGuAGcAGcAGTsT AD-12768 b 633-651 413 cuGcuGcuAcuAuAGAAGuTsT 414 AcUUCuAuAGuAGcAGcAGTsT AD-12673 a 634-652 415 uGcuGcuAcuAuAGAAGuuTsT 416 AaCUUCuAuAGuAGcAGcATsT AD-12769 b 634-652 417 uGcuGcuAcuAuAGAAGuuTsT 418 AACUUCuAuAGuAGcAGcATsT AD-12823 c 634-652 419 ugcuGcuAcuAuAGAAGuuTsT 420 AACUUCuAuAGuAGcAGcATsT AD-12864 d 634-652 421 ugcuGcuAcuAuAGAAGuuTsT 422 AaCUUCuAuAGuAGcAGcATsT AD-12674 a 635-653 423 GcuGcuAcuAuAGAAGuuGTsT 424 caACUUCuAuAGuAGcAGCTsT AD-12770 b 635-653 425 GcuGcuAcuAuAGAAGuuGTsT 426 cAACUUCuAuAGuAGcAGCTsT AD-12675 a 636-654 427 cuGcuAcuAuAGAAGuuGATsT 428 UcAAcUUCuAuAGuAGcAGTsT AD-12676 a 637-655 429 uGcuAcuAuAGAAGuuGAATsT 430 UucAACUUCuAuAGuAGcATsT AD-12771 b 637-655 431 uGcuAcuAuAGAAGuuGAATsT 432 UUcAACUUCuAuAGuAGcATsT AD-12824 c 637-655 433 ugcuAcuAuAGAAGuuGAATsT 434 UUcAACUUCuAuAGuAGcATsT AD-12865 d 637-655 435 ugcuAcuAuAGAAGuuGAATsT 436 UucAACUUCuAuAGuAGcATsT AD-12677 a 912-930 437 cAGAAGAcuAcuAuGAuAuTsT 438 AuAUcAuAGuAGUCUUCUGTsT AD-12825 c 912-930 439 caGAAGAcuAcuAuGAuAuTsT 440 AuAUcAuAGuAGUCUUCUGTsT AD-12678 a 4153-4171 441 AAAuuuuAuAuAAGAAAcuTsT 442 AgUUUCUuAuAuAAAAUUUTsT AD-12772 b 4153-4171 443 AAAuuuuAuAuAAGAAAcuTsT 444 AgUUUCUuAuAuAAAAUUUTsT AD-12826 c 4153-4171 445 AaAuuuuAuAuAAGAAAcuTsT 446 AGUUUCUuAuAuAAAAUUUTsT AD-12866 d 4153-4171 447 AaAuuuuAuAuAAGAAAcuTsT 448 AGUUUCUuAuAuAAAAUUUTsT AD-12679 a 4779-4797 449 AuGGAAuAGuucAGAGGuuTsT 450 AaCCUCUGAACuAUUCcAUTsT AD-12773 b 4779-4797 451 AuGGAAuAGuucAGAGGuuTsT 452 AACCUCUGAACuAUUCcAUTsT AD-12680 a 4779-4797 453 cAuGGAAuAGuucAGAGGuTsT 454 AcCUCUGAACuAUUCcAUGTsT AD-12774 b 4779-4797 455 cAuGGAAuAGuucAGAGGuTsT 456 ACCUCUGAACuAUUCcAUGTsT AD-12827 c 4779-4797 457 cauGGAAuAGuucAGAGGuTsT 458 ACCUCUGAACuAUUCcAUGTsT AD-12867 d 4779-4797 459 cauGGAAuAGuucAGAGGuTsT 460 AcCUCUGAACuAUUCcAUGTsT AD-12681 a 4781-4799 461 AcAuGGAAuAGuucAGAGGTsT 462 CcUCUGAACuAUUCcAUGUTsT AD-12775 b 4781-4799 463 AcAuGGAAuAGuucAGAGGTsT 464 CCUCUGAACuAUUCcAUGUTsT AD-12682 a 4784-4802 2465 GGuAcAuGGAAuAGuucAGTsT 466 CuGAACuAUUCcAUGuACCTsT AD-12776 b 4784-4802 2467 GGuAcAuGGAAuAGuucAGTsT 468 CUGAACuAUUCcAUGuACCTsT AD-12828 c 4784-4802 2469 GguAcAuGGAAuAGuucAGTsT 470 CUGAACuAUUCcAUGuACCTsT AD-12868 d 4784-4802 2471 GguAcAuGGAAuAGuucAGTsT 472 CuGAACuAUUCcAUGuACCTsT AD-12683 a 4785-4803 473 uGGuAcAuGGAAuAGuucATsT 474 UgAACuAUUCcAUGuACcATsT AD-12777 b 4785-4803 475 uGGuAcAuGGAAuAGuucATsT 476 UGAACuAUUCcAUGuACcATsT AD-12829 c 4785-4803 477 ugGuAcAuGGAAuAGuucATsT 478 UGAACuAUUCcAUGuACcATsT AD-12869 d 4785-4803 479 ugGuAcAuGGAAuAGuucATsT 480 UgAACuAUUCcAUGuACcATsT AD-12684 a 719-737 481 cuuAcuccuGAAAcAuAuGTsT 482 cauAUGUUUcAGGAGuAAGTsT AD-12778 b 719-737 483 cuuAcuccuGAAAcAuAuGTsT 484 cAuAUGUUUcAGGAGuAAGTsT AD-12685 a 909-927 485 AuccAGAAGAcuAcuAuGATsT 486 UcAuAGuAGUCUUCUGGAUTsT AD-12686 a 1119-1137 487 uuuuGGAAGAAAcuAcuuGTsT 488 caAGuAGUUUCUUCcAAAATsT AD-12779 b 1119-1137 489 uuuuGGAAGAAAcuAcuuGTsT 490 cAAGuAGUUUCUUCcAAAATsT AD-12687 a 1121-1139 491 uuGGAAGAAAcuAcuuGGGTsT 492 CccAAGuAGUUUCUUUCcAATsT AD-12780 b 1121-1139 493 uuGGAAGAAAcuAcuuGGGTsT 494 CCcAAGuAGUUUCUUUCcAATsT AD-12688 a 4357-4375 495 AuGAAGAccuGuuuuGccATsT 496 UgGcAAAAcAGGUCUUcAUTsT AD-12781 b 4357-4375 497 AuGAAGAccuGuuuuGccATsT 498 UGGcAAAAcAGGUCUUcAUTsT AD-12689 a 4358-4376 499 GAuGAAGAccuGuuuuGccTsT 500 GgcAAAAcAGGUCUUcAUCTsT AD-12782 b 4358-4376 501 GAuGAAGAccuGuuuuGccTsT 502 GGcAAAAcAGGUCUUcAUCTsT AD-12830 c 4358-4376 503 GauGAAGAccuGuuuuGccTsT 504 GGcAAAAcAGGUCUUcAUCTsT AD-12870 d 4358-4376 505 GauGAAGAccuGuuuuGccTsT 506 GgcAAAAcAGGUCUUcAUCTsT AD-12690 a 4360-4378 507 GGGAuGAAGAccuGuuuuGTsT 508 caAAAcAGGUCUUcAUCCCTsT AD-12783 b 4360-4378 509 GGGAuGAAGAccuGuuuuGTsT 510 cAAAAcAGGUCUUcAUCCCTsT AD-12831 c 4360-4378 511 GgGAuGAAGAccuGuuuuGTsT 512 cAAAAcAGGUCUUcAUCCCTsT AD-12871 d 4360-4378 513 GgGAuGAAGAccuGuuuuGTsT 514 caAAAcAGGUCUUcAUCCCTsT exo/endo-light + 2′-O-methyl in position 2 of antisense exo/endo-light: sense strand: dTsdT + 2′OME@all Py; antinsense strand: dTsdT + 2′OMe@ Py in uA, cA exo/endo-light + 2′O-methyl in position 2 of sense exo/endo-light + 2′O-methyl in position 2 of sense and antisense

Residual luciferase Relative activity siRNA (relative to SD of activity control residual Residual (normalized SD of Relative siRNA luci- luciferase to positive relative siRNA treated ferase activity +/− control luc- siRNA activity +/− cells) activity SD siRNA) activity SD 91 11  91 ± 11% 9 2 9 ± 2% 32 5 32 ± 5% 76 17 76 ± 17% 25 6 25 ± 6% 79 13 79 ± 13% 16 4 16 ± 4% 97 26 97 ± 26% 79 9 79 ± 9% 21 3 21 ± 3%  25 4 25 ± 4% 85 24 85 ± 24% 23 2 23 ± 2% 87 14 87 ± 14% 84 11  84 ± 11% 18 4 18 ± 4%  102 8 102 ± 8%  −6 1 −6 ± 1%  95 10  95 ± 10% 6 1 6 ± 1% 107 9 107 ± 9%  −11 2 −11 ± 2%  70 4 70 ± 4% 34 3 34 ± 3%  69 8 69 ± 8% 35 7 35 ± 7%  94 8 94 ± 8% 7 1 7 ± 1% 100 9 100 ± 9%  −4 1 −4 ± 1%  27 5 27 ± 5% 82 16 82 ± 16% 15 2 15 ± 2% 94 13 94 ± 13% 94 5 94 ± 5% 7 0 7 ± 0% 61 10  61 ± 10% 41 8 41 ± 8%  55 6 55 ± 6% 47 6 47 ± 6%  92 16  92 ± 16% 8 2 8 ± 2% 78 3 78 ± 3% 25 1 25 ± 1%  63 6 63 ± 6% 42 5 42 ± 5%  101 9 101 ± 9%  −1 0 −1 ± 0%  101 5 101 ± 5%  −1 0 −1 ± 0%  85 18  85 ± 18% 15 4 15 ± 4%  95 9 95 ± 9% 6 1 6 ± 1% 103 13 103 ± 13% −3 0 −3 ± 0%  81 9 81 ± 9% 22 3 22 ± 3%  61 4 61 ± 4% 44 4 44 ± 4%  103 11 103 ± 11% −3 0 −3 ± 0%  108 19 108 ± 19% −9 2 −9 ± 2%  94 17  94 ± 17% 7 1 7 ± 1% 88 9 88 ± 9% 14 2 14 ± 2%  39 4 39 ± 4% 64 8 64 ± 8%  38 6 38 ± 6% 69 12 69 ± 12% 26 4 26 ± 4% 78 13 78 ± 13% 17 3 17 ± 3% 87 18 87 ± 18% 22 4 22 ± 4% 81 16 81 ± 16% 100 6 100 ± 6%  0 0 0 ± 0% 73 6 73 ± 6% 28 3 28 ± 3%  46 9 46 ± 9% 57 12 57 ± 12% 97 15  97 ± 15% 3 1 3 ± 1% 26 4 26 ± 4% 82 15 82 ± 15% 10 1 10 ± 1% 94 12 94 ± 12% 10 3 10 ± 3% 94 40 94 ± 40% 22 3 22 ± 3% 81 12 81 ± 12% 15 5 15 ± 5% 94 38 94 ± 38% 6 1  6 ± 1% 98 26 98 ± 26% 93 4 93 ± 4% 8 0 8 ± 0% 95 4 95 ± 4% 5 0 5 ± 0% 73 7 73 ± 7% 30 3 30 ± 3%  88 10  88 ± 10% 13 2 13 ± 2%  42 7 42 ± 7% 60 7 60 ± 7%  21 5 21 ± 5% 89 32 89 ± 32% 95 7 95 ± 7% 6 1 6 ± 1% 71 2 71 ± 2% 30 1 30 ± 1%  54 7 54 ± 7% 48 7 48 ± 7%  94 9 94 ± 9% 7 1 7 ± 1% 106 7 106 ± 7%  −8 1 −8 ± 1%  100 7 100 ± 7%  0 0 0 ± 0% 107 9 107 ± 9%  −7 1 −7 ± 1%  47 4 47 ± 4% 60 8 60 ± 8%  40 8 40 ± 8% 67 20 67 ± 20% 78 13  78 ± 13% 25 8 25 ± 8%  16 4 16 ± 4% 92 29 92 ± 29% 25 6 25 ± 6% 84 29 84 ± 29% 23 3 23 ± 3% 86 20 86 ± 20% 19 4 19 ± 4% 91 20 91 ± 20% 103 9 103 ± 9%  −3 0 −3 ± 0%  84 8 84 ± 8% 17 2 17 ± 2%  31 4 31 ± 4% 77 12 77 ± 12% 18 1 18 ± 1% 85 3 85 ± 3%  94 10  94 ± 10% 5 1 5 ± 1% 57 4 57 ± 4% 48 4 48 ± 4%  99 7 99 ± 7% 0 0 0 ± 0% 82 5 82 ± 5% 20 1 20 ± 1%  80 6 80 ± 6% 22 2 22 ± 2%  65 6 65 ± 6% 39 4 39 ± 4%  81 6 81 ± 6% 21 2 21 ± 2%  82 7 82 ± 7% 21 2 21 ± 2%  113 11 113 ± 11% −14 2 −14 ± 2%  90 9 90 ± 9% 11 1 11 ± 1%  92 8 92 ± 8% 9 1 9 ± 1% 117 7 117 ± 7%  −19 1 −19 ± 1%  124 3 124 ± 3%  −27 1 −27 ± 1%  85 4 85 ± 4% 16 1 16 ± 1%  52 1 52 ± 1% 53 1 53 ± 1%  96 4 96 ± 4% 5 0 5 ± 0% 110 11 110 ± 11% −12 1 −12 ± 1%  115 13 115 ± 13% −17 2 −17 ± 2%  106 2 106 ± 2%  −7 0 −7 ± 0%  107 12 107 ± 12% −8 1 −8 ± 1%  88 5 88 ± 5% 14 1 14 ± 1%  79 5 79 ± 5% 24 1 24 ± 1%  69 8 69 ± 8% 35 6 35 ± 6%  75 8 75 ± 8% 25 6 25 ± 6%  65 8 65 ± 8% 40 8 40 ± 8%  56 4 56 ± 4% 50 6 50 ± 6%  74 6 74 ± 6% 30 3 30 ± 3%  89 8 89 ± 8% 9 1 9 ± 1% 31 4 31 ± 4% 78 14 78 ± 14% 16 2 16 ± 2% 93 14 93 ± 14% 18 3 18 ± 3% 85 14 85 ± 14% 18 4 18 ± 4% 86 22 86 ± 22% 15 2 15 ± 2% 89 14 89 ± 14% 95 6 95 ± 6% 5 0 5 ± 0% 23 4 23 ± 4% 81 15 81 ± 15% 14 1 14 ± 1% 90 10 90 ± 10% 90 12  90 ± 12% 10 2 10 ± 2%  113 11 113 ± 11% −15 2 −15 ± 2%  42 4 42 ± 4% 60 7 60 ± 7%  34 3 34 ± 3% 68 8 68 ± 8%  114 3 114 ± 3%  −14 0 −14 ± 0%  96 11  96 ± 11% 4 1 4 ± 1% 52 7 52 ± 7% 53 8 53 ± 8%  74 9 74 ± 9% 29 4 29 ± 4%  111 5 111 ± 5%  −12 1 −12 ± 1%  103 8 103 ± 8%  −3 0 −3 ± 0%  94 13  94 ± 13% 6 1 6 ± 1% 105 3 105 ± 3%  −6 0 −6 ± 0%  100 9 100 ± 9%  0 0 0 ± 0% 33 4 33 ± 4% 74 10 74 ± 10% 21 3 21 ± 3% 83 13 83 ± 13% 25 4 25 ± 4% 78 14 78 ± 14% 28 4 28 ± 4% 75 11 75 ± 11% 82 7 82 ± 7% 20 2 20 ± 2%  25 4 25 ± 4% 78 14 78 ± 14% 23 7 23 ± 7% 80 30 80 ± 30% 61 7 61 ± 7% 41 5 41 ± 5%  112 6 112 ± 6%  −14 1 −14 ± 1%  86 10  86 ± 10% 16 2 16 ± 2%  94 10  94 ± 10% 6 1 6 ± 1% 93 11  93 ± 11% 7 1 7 ± 1% 77 8 77 ± 8% 24 3 24 ± 3%  96 4 96 ± 4% 5 0 5 ± 0% 27 3 27 ± 3% 81 11 81 ± 11% 29 6 29 ± 6% 74 19 74 ± 19% 31 2 31 ± 2% 72 6 72 ± 6%  26 3 26 ± 3% 78 11 78 ± 11% 81 9 81 ± 9% 17 3 17 ± 3%  92 9 92 ± 9% 8 1 8 ± 1% 71 9 71 ± 9% 30 5 30 ± 5%  81 2 81 ± 2% 21 1 21 ± 1%  57 1 57 ± 1% 48 1 48 ± 1%  52 4 52 ± 4% 54 5 54 ± 5%  77 5 77 ± 5% 26 2 26 ± 2%  89 6 89 ± 6% 13 1 13 ± 1%  88 7 88 ± 7% 12 1 12 ± 1%  67 6 67 ± 6% 35 4 35 ± 4%  88 10  88 ± 10% 12 2 12 ± 2%  91 2 91 ± 2% 10 0 10 ± 0%  40 3 40 ± 3% 67 6 67 ± 6%  35 1 35 ± 1% 72 3 72 ± 3%  75 8 75 ± 8% 28 4 28 ± 4%  79 8 79 ± 8% 23 3 23 ± 3%  17 5 17 ± 5% 86 27 86 ± 27% 97 6 97 ± 6% 3 0 3 ± 0% 74 5 74 ± 5% 27 2 27 ± 2%  46 6 46 ± 6% 59 9 59 ± 9%  14 0 14 ± 0% 89 2 89 ± 2%  12 3 12 ± 3% 92 28 92 ± 28% 35 7 35 ± 7% 70 17 70 ± 17% 10 3 10 ± 3% 99 33 99 ± 33% 9 1  9 ± 1% 95 18 95 ± 18% 108 1 108 ± 1%  −9 0 −9 ± 0%  101 4 101 ± 4%  −1 0 −1 ± 0%  98 9 98 ± 9% 2 0 2 ± 0% 83 4 83 ± 4% 18 1 18 ± 1%  80 14  80 ± 14% 21 4 21 ± 4%  25 3 25 ± 3% 79 11 79 ± 11% 67 4 67 ± 4% 35 2 35 ± 2%  95 11  95 ± 11% 8 3 8 ± 3% 66 7 66 ± 7% 39 6 39 ± 6%  34 2 34 ± 2% 73 5 73 ± 5%  10 3 10 ± 3% 94 30 94 ± 30% 12 4 12 ± 4% 92 37 92 ± 37% 33 1 33 ± 1% 72 2 72 ± 2%  92 7 92 ± 7% 7 1 7 ± 1% 91 11  91 ± 11% 10 2 10 ± 2%  99 10  99 ± 10% 3 1 3 ± 1% 20 5 20 ± 5% 89 22 89 ± 22% 20 4 20 ± 4% 90 20 90 ± 20% 93 11  93 ± 11% 8 1 8 ± 1% 93 9 93 ± 9% 6 2 6 ± 2% 94 8 94 ± 8% 10 1 10 ± 1%  58 8 58 ± 8% 47 10 47 ± 10% 49 6 49 ± 6% 58 9 58 ± 9%  93 8 93 ± 8% 8 1 8 ± 1% 30 5 30 ± 5% 76 18 76 ± 18% 25 2 25 ± 2% 84 9 84 ± 9%  65 10  65 ± 10% 38 7 38 ± 7%  34 7 34 ± 7% 69 17 69 ± 17% 34 4 34 ± 4% 73 10 73 ± 10% 13 3 13 ± 3% 91 22 91 ± 22% 11 2 11 ± 2% 93 17 93 ± 17% 19 4 19 ± 4% 87 22 87 ± 22% 22 3 22 ± 3% 87 12 87 ± 12% 11 4 11 ± 4% 93 39 93 ± 39% 45 3 45 ± 3% 61 5 61 ± 5%  10 3 10 ± 3% 94 31 94 ± 31% 12 1 12 ± 1% 92 13 92 ± 13% 41 4 41 ± 4% 64 8 64 ± 8%  83 9 83 ± 9% 19 2 19 ± 2%  74 7 74 ± 7% 29 3 29 ± 3%  52 7 52 ± 7% 54 9 54 ± 9%  28 3 28 ± 3% 81 12 81 ± 12% 56 5 56 ± 5% 49 5 49 ± 5%  36 2 36 ± 2% 72 5 72 ± 5%  33 2 33 ± 2% 75 5 75 ± 5%  49 7 49 ± 7% 57 10 57 ± 10% 90 9 90 ± 9% 11 1 11 ± 1%  45 6 45 ± 6% 61 9 61 ± 9%  45 5 45 ± 5% 62 8 62 ± 8%  47 6 47 ± 6% 59 9 59 ± 9%  31 4 31 ± 4% 77 11 77 ± 11% 31 3 31 ± 3% 77 10 77 ± 10% 43 7 43 ± 7% 64 12 64 ± 12% 23 4 23 ± 4% 86 16 86 ± 16% 22 4 22 ± 4% 87 16 87 ± 16% 102 8 102 ± 8%  −2 0 −2 ± 0%  101 13 101 ± 13% −1 0 −1 ± 0%  99 1 99 ± 1% 1 0 1 ± 0% 91 7 91 ± 7% 10 1 10 ± 1%  81 8 81 ± 8% 21 2 21 ± 2%  11 2 11 ± 2% 93 19 93 ± 19% 17 3 17 ± 3% 92 17 92 ± 17% 15 2 15 ± 2% 89 17 89 ± 17% 11 2 11 ± 2% 93 18 93 ± 18% 15 3 15 ± 3% 91 22 91 ± 22% 28 3 28 ± 3% 79 10 79 ± 10% 8 1  8 ± 1% 95 19 95 ± 19% 43 6 43 ± 6% 63 9 63 ± 9%  23 5 23 ± 5% 80 19 80 ± 19% 23 5 23 ± 5% 80 20 80 ± 20% 25 4 25 ± 4% 81 16 81 ± 16% 17 2 17 ± 2% 91 15 91 ± 15% 11 2 11 ± 2% 92 22 92 ± 22% 12 1 12 ± 1% 92 11 92 ± 11% 19 3 19 ± 3% 87 16 87 ± 16% 87 12  87 ± 12% 14 2 14 ± 2%  41 4 41 ± 4% 66 8 66 ± 8%  35 1 35 ± 1% 72 1 72 ± 1%  68 5 68 ± 5% 36 3 36 ± 3%  58 5 58 ± 5% 47 5 47 ± 5%  73 8 73 ± 8% 30 4 30 ± 4%  62 8 62 ± 8% 42 7 42 ± 7%  18 1 18 ± 1% 91 4 91 ± 4%  11 3 11 ± 3% 93 33 93 ± 33% 96 4 96 ± 4% 4 0 4 ± 0% 45 7 45 ± 7% 58 10 58 ± 10% 15 3 15 ± 3% 89 19 89 ± 19% 51 3 51 ± 3% 52 4 52 ± 4%  93 6 93 ± 6% 8 1 8 ± 1% 36 3 36 ± 3% 66 7 66 ± 7%  27 2 27 ± 2% 76 7 76 ± 7%  81 18  81 ± 18% 21 5 21 ± 5%  

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human JC virus genome in a cell, wherein said dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding JC virus and wherein said region of complementarity is less that 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said JC virus, inhibits expression of said JC virus genome.
 2. The dsRNA of claim 1, wherein said first sequence is selected from the group consisting of Tables 1a and b and said second sequence is selected from the group consisting of Tables 1a and b.
 3. The dsRNA of claim 1, wherein said dsRNA comprises at least one modified nucleotide.
 4. The dsRNA of claim 2, wherein said dsRNA comprises at least one modified nucleotide.
 5. The dsRNA of claims 3 or 4, wherein said modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
 6. The dsRNA of claims 3 or 4, wherein said modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, and abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
 7. The dsRNA of claims 3 or 4, wherein said first sequence is selected from the group consisting of Tables 1a and b and said second sequence is selected from the group consisting of Tables 1a and b.
 8. The dsRNA of claims 6 or 7, wherein said first sequence is selected from the group consisting of Tables 1a and b and said second sequence is selected from the group consisting of Tables 1a and b.
 9. A cell comprising the dsRNA of claim
 1. 10. A pharmaceutical composition for inhibiting the expression of a gene from the JC Virus in an organism, comprising a dsRNA and a pharmaceutically acceptable carrier, wherein the dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding JC virus, and wherein said region of complementarity is less tan 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said JC virus, inhibits expression of said JC virus genome.
 11. The pharmaceutical composition of claim 10, wherein said first sequence of said dsRNA is selected from the group consisting of Tables 1a Laid b aid said second sequence of said dsRNA is selected from the group consisting of Tables 1a and b.
 12. The pharmaceutical composition of claim 10, wherein said first sequence of said dsRNA is selected from the group consisting of Tables 1a and b and said second sequence of said dsRNA is selected from the group consisting of Tables 1a and b.
 13. A method for inhibiting the expression of a gene from the JC Virus in a cell, the method comprising: (a) introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region or complementarity which is substantially complementary to at least a par of a mRNA encoding JC virus, and wherein said region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said JC virus inhibits expression of said JC virus genome; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a gene from the JC Virus, thereby inhibiting expression of a gene from the JC Virus in the cell.
 14. A method of treating, preventing or managing pathological processes medicated by JC virus expression comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of a dsRNA, wherein the dsRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding JC virus, and wherein and region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said JC virus, inhibits expression of said JC virus genome.
 15. A vector for inhibiting the expression of a gene from the JC Virus in a cell, said vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least on strand of a dsRNA, wherein one of the strands of said dsRNA is substantially complementary to at least a part of a mRNA encoding JC virus and wherein said dsRNA is less than 30 base pairs in length and wherein said dsRNA, upon contact with a cell expressing said JC virus, inhibits the expression of said JC virus genome.
 16. A cell comprising the vector of claim
 15. 